Compositions and methods for silencing scn9a expression

ABSTRACT

The disclosure relates to double-stranded ribonucleic acid (dsRNA) compositions targeting SCN9A, and methods of using such dsRNA compositions to alter (e.g., inhibit) expression of SCN9A.

RELATED APPLICATIONS

This application claims priority to U.S. provisional application No. 63/006,328, filed on Apr. 7, 2020, and U.S. provisional application No. 63/161,313, filed on Mar. 15, 2021. The entire contents of the foregoing applications are hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 2, 2021, is named A2038-7235WO_SL.txt and is 1,514,568 bytes in size.

FIELD OF THE DISCLOSURE

The disclosure relates to the specific inhibition of the expression of the SCN9A gene.

BACKGROUND

Pain, e.g., chronic pain is a prevalent symptom and major cause of disability. Chronic pain can result from inflammatory pain or neuropathic pain, or it can be associated with a disease or disorder, e.g., cancer, arthritis, diabetes, traumatic injury and/or viral infections. Hypersensitivity or hyposensitivity to pain can also result from pain-related disorders, including but not limited to an inability to sense pain, primary erythromelalgia (PE), and paroxysmal extreme pain disorder (PEPD). Current therapies for pain are non-selective for their targets and result in unwanted, off-target effects involving the central nervous system (CNS). New treatments for pain, e.g., chronic pain and pain-related disorders are needed.

SUMMARY

The present disclosure describes methods and iRNA compositions for modulating the expression of SCN9A. In certain embodiments, expression of SCN9A is reduced or inhibited using an SCN9A-specific iRNA. Such inhibition can be useful in treating disorders related to SCN9A expression, such as pain, e.g., acute pain or chronic pain (e.g., inflammatory pain, neuropathic pain, pain hypersensitivity, pain hyposensitivity, inability to sense pain, primary erythromelalgia (PE), paroxysmal extreme pain disorder (PEPD), small fiber neuropathy (SFN), trigeminal neuralgia (TN) and pain associated with e.g., cancer, arthritis, diabetes, traumatic injury and viral infections).

Accordingly, described herein are compositions and methods that effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of SCN9A, such as in a cell or in a subject (e.g., in a mammal, such as a human subject). Also described are compositions and methods for treating a disorder related to expression of SCN9A, such as pain (e.g., acute pain or chronic pain, e.g., inflammatory pain, neuropathic pain, pain hypersensitivity, pain hyposensitivity, inability to sense pain, primary erythromelalgia (PE), paroxysmal extreme pain disorder (PEPD), small fiber neuropathy (SFN), trigeminal neuralgia (TN) and pain associated with, e.g., cancer, arthritis, diabetes, traumatic injury and viral infections).

The iRNAs (e.g., dsRNAs) included in the compositions featured herein include an RNA strand (the antisense strand) having a region, e.g., a region that is 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of SCN9A (e.g., a human SCN9A) (also referred to herein as an “SCN9A-specific iRNA”). In some embodiments, the SCN9A mRNA transcript is a human SCN9A mRNA transcript, e.g., SEQ ID NO: 1 herein.

In some embodiments, the iRNA (e.g., dsRNA) described herein comprises an antisense strand having a region that is substantially complementary to a region of a human SCN9A mRNA. In some embodiments, the human SCN9A mRNA has the sequence NM_002977.3 (SEQ ID NO: 1) or NM_001365536.1 (SEQ ID NO: 4001). In some embodiments, the human SCN9A mRNA has the sequence NM_002977.3 (SEQ ID NO: 1). The sequence of NM_002977.3 is also herein incorporated by reference in its entirety. The reverse complement of SEQ ID NO: 1 is provided as SEQ ID NO: 2 herein. In some embodiments, the human SCN9A mRNA has the sequence NM_001365536.1 (SEQ ID NO: 4001). The sequence of NM_001365536.1 is also herein incorporated by reference in its entirety. The reverse complement of SEQ ID NO: 4001 is provided as SEQ ID NO: 4002 herein.

In some aspects, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of sodium channel, voltage gated, type IX alpha subunit (SCN9A), wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of a coding strand of human SCN9A and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of a non-coding strand of human SCN9A such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.

In some aspects, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of SCN9A, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.

In some aspects, the present disclosure provides a human cell or tissue comprising a reduced level of SCN9A mRNA or a level of SCN9A protein as compared to an otherwise similar untreated cell or tissue, wherein optionally the cell or tissue is not genetically engineered (e.g., wherein the cell or tissue comprises one or more naturally arising mutations, e.g., SCN9A), wherein optionally the level is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the human cell or tissue is a human peripheral sensory neuron (e.g., a peripheral sensory neuron in a dorsal root ganglion, or a nociceptive neuron, e.g., an A-delta fiber or a C-type fiber).

The present disclosure also provides, in some aspects, a cell containing the dsRNA agent described herein.

In some aspects, the present disclosure also provides a pharmaceutical composition for inhibiting expression of a gene encoding SCN9A, comprising a dsRNA agent described herein.

The present disclosure also provides, in some aspects, a method of inhibiting expression of SCN9A in a cell, the method comprising:

(a) contacting the cell with the dsRNA agent described herein, or a pharmaceutical composition described herein; and

(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of SCN9A, thereby inhibiting expression of the SCN9A in the cell.

The present disclosure also provides, in some aspects, a method of inhibiting expression of SCN9A in a cell, the method comprising:

(a) contacting the cell with the dsRNA agent described herein, or a pharmaceutical composition described herein; and

(b) maintaining the cell produced in step (a) for a time sufficient to reduce levels of SCN9A mRNA, SCN9A protein, or both of SCN9A mRNA and protein, thereby inhibiting expression of the SCN9A in the cell.

The present disclosure also provides, in some aspects, a method of inhibiting expression of SCN9A in a cell or a tissue of the central nervous system (CNS), the method comprising:

(a) contacting the cell or tissue with a dsRNA agent that binds SCN9A; and

(b) maintaining the cell or tissue produced in step (a) for a time sufficient to reduce levels of SCN9A mRNA, SCN9A protein, or both of SCN9A mRNA and protein, thereby inhibiting expression of SCN9A in the cell or tissue.

The present disclosure also provides, in some aspects, a method of treating a subject diagnosed with SCN9A-associated disorder comprising administering to the subject a therapeutically effective amount of the dsRNA agent described herein or a pharmaceutical composition described herein, thereby treating the disorder.

In any of the aspects herein, e.g., the compositions and methods above, any of the embodiments herein (e.g., below) may apply.

In some embodiments, the coding strand of human SCN9A has the sequence of SEQ ID NO: 1. In some embodiments, the non-coding strand of human SCN9A has the sequence of SEQ ID NO: 2. In some embodiments, the coding strand of human SCN9A has the sequence of SEQ ID NO: 4001. In some embodiments, the non-coding strand of human SCN9A has the sequence of SEQ ID NO: 4002.

In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 4001.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 17 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 4002 such that the sense strand is complementary to the at least 17 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 4001.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 19 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 4002 such that the sense strand is complementary to the at least 19 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 4001.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 21 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 4002 such that the sense strand is complementary to the at least 21 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 4001.

In some embodiments, the portion of the sense strand is a portion within nucleotides 581-601, 760-780, or 8498-8518 of SEQ ID NO: 4001. In some embodiments, the portion of the sense strand is a portion corresponding to SEQ ID NO: 4827, 5026, or 4822.

In some embodiments, the portion of the sense strand is a portion within a sense strand in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20.

In some embodiments, the portion of the antisense strand is a portion within an antisense strand in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20.

In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 that corresponds to the antisense sequence.

In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 that corresponds to the antisense sequence.

In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 that corresponds to the antisense sequence.

In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 that corresponds to the antisense sequence.

In some embodiments, the sense strand of the dsRNA agent is at least 23 nucleotides in length, e.g., 23-30 nucleotides in length.

In some embodiments, the portion of the sense strand is a portion within a sense strand from a duplex chosen from AD-1251284 (UGUCGAGUACACUUUUACUGA (SEQ ID NO:4827)), AD-961334 (CAACACAATUTCUUCUUAGCA (SEQ ID NO: 5026)), or AD-1251325 (AAAACAAUCUUCCGUUUCAAA (SEQ ID NO: 4822)). In some embodiments, the portion is a portion of a corresponding chemically modified sequence provided in Tables 5A, 13A, 14A, 15A, and 16.

In some embodiments, the portion of the sense strand is a sense strand chosen from the sense strands of AD-1251284 (UGUCGAGUACACUUUUACUGA (SEQ ID NO:4827)), AD-961334 (CAACACAATUTCUUCUUAGCA (SEQ ID NO: 5026)), or AD-1251325 (AAAACAAUCUUCCGUUUCAAA (SEQ ID NO: 4822)). In some embodiments, the portion is a portion of a corresponding chemically modified sequence provided in Tables 5A, 13A, 14A, 15A, and 16.

In some embodiments, the portion of the antisense strand is a portion within an antisense strand from a duplex chosen from AD-1251284 (UCAGTAAAAGUGUACTCGACAUU (SEQ ID NO: 5093)), AD-961334 (UGCUAAGAAGAAATUGUGUUGUU (SEQ ID NO: 5292)), or AD-1251325 (UUUGAAACGGAAGAUUGUUUUCC (SEQ ID NO: 5088)). In some embodiments, the portion is a portion of a corresponding chemically modified sequence provided in Tables 5A, 13A, 14A, 15A, and 16.

In some embodiments, the portion of the antisense strand is an antisense strand chosen from the antisense strands of AD-1251284 (UCAGTAAAAGUGUACTCGACAUU (SEQ ID NO: 5093)), AD-961334 (UGCUAAGAAGAAATUGUGUUGUU (SEQ ID NO: 5292)), or AD-1251325 (UUUGAAACGGAAGAUUGUUUUCC (SEQ ID NO: 5088)). In some embodiments, the portion is a portion of a corresponding chemically modified sequence provided in Tables 5A, 13A, 14A, 15A, and 16.

In some embodiments, the sense strand and the antisense strand of the dsRNA agent comprise nucleotide sequences of the paired sense strand and antisense strand of a duplex selected from AD-1251284 (SEQ ID NO: 4827 and 5093), AD-961334 (SEQ ID NO: 5026 and 5292), or AD-1251325 (SEQ ID NO: 4822 and 5088). In some embodiments, the sense strand and antisense strand comprises the corresponding chemically modified sense sequence and antisense sequence provided in Tables 5A, 13A, 14A, 15A, and 16.

In some embodiments, at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties. In some embodiments, the lipophilic moiety is conjugated to one or more positions in the double stranded region of the dsRNA agent. In some embodiments, the lipophilic moiety is conjugated via a linker or carrier. In some embodiments, lipophilicity of the lipophilic moiety, measured by logKow, exceeds 0. In some embodiments,

In some embodiments, the hydrophobicity of the double-stranded RNAi agent, measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent, exceeds 0.2. In some embodiments, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

In some embodiments, the dsRNA agent comprises at least one modified nucleotide. In some embodiments, no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand are unmodified nucleotides. In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In some embodiments, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxythimidine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a glycol modified nucleotide, and a 2-O—(N-methylacetamide) modified nucleotide; and combinations thereof. In some embodiments, no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand include modifications other than 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA).

In some embodiments, the dsRNA comprises a non-nucleotide spacer (wherein optionally the non-nucleotide spacer comprises a C3-C6 alkyl) between two of the contiguous nucleotides of the sense strand or between two of the contiguous nucleotides of the antisense strand.

In some embodiments, each strand is no more than 30 nucleotides in length. In some embodiments, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In some embodiments, at least one strand comprises a 3′ overhang of at least 2 nucleotides. In some embodiments, at least one strand comprises a 3′ overhang of 2 nucleotides.

In some embodiments, the double stranded region is 15-30 nucleotide pairs in length. In some embodiments, the double stranded region is 17-23 nucleotide pairs in length. In some embodiments, the double stranded region is 17-25 nucleotide pairs in length. In some embodiments, the double stranded region is 23-27 nucleotide pairs in length. In some embodiments, the double stranded region is 19-21 nucleotide pairs in length. In some embodiments, the double stranded region is 21-23 nucleotide pairs in length. In some embodiments, each strand has 19-30 nucleotides. In some embodiments, each strand has 19-23 nucleotides. In some embodiments, each strand has 21-23 nucleotides.

In some embodiments, the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand. In some embodiments, the strand is the antisense strand. In some embodiments, the strand is the sense strand.

In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand. In some embodiments, the strand is the antisense strand. In some embodiments, the strand is the sense strand.

In some embodiments, each of the 5′- and 3′-terminus of one strand comprises a phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the strand is the antisense strand.

In some embodiments, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand. In some embodiments, the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via a linker or carrier.

In some embodiments, the internal positions include all positions except the terminal two positions from each end of the at least one strand. In some embodiments, the internal positions include all positions except the terminal three positions from each end of the at least one strand. In some embodiments, the internal positions exclude a cleavage site region of the sense strand. In some embodiments, the internal positions include all positions except positions 9-12, counting from the 5′-end of the sense strand. In some embodiments, the internal positions include all positions except positions 11-13, counting from the 3′-end of the sense strand. In some embodiments, the internal positions exclude a cleavage site region of the antisense strand. In some embodiments, the internal positions include all positions except positions 12-14, counting from the 5′-end of the antisense strand. In some embodiments, the internal positions include all positions except positions 11-13 on the sense strand, counting from the 3′-end, and positions 12-14 on the antisense strand, counting from the 5′-end.

In some embodiments, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′end of each strand. In some embodiments, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5′-end of each strand.

In some embodiments, the positions in the double stranded region exclude a cleavage site region of the sense strand.

In some embodiments, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand. In some embodiments, the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 21, position 20, or position 15 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 16 of the antisense strand. In some embodiments, the lipophilic moiety is conjugated to position 6, counting from the 5′-end of the sense strand.

In some embodiments, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound. In some embodiments, the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

In some embodiments, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region. In some embodiments, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

In some embodiments, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.

In some embodiments, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.

In some embodiments, the lipophilic moiety or targeting ligand is conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

In some embodiments, the 3′ end of the sense strand is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

In some embodiments, the dsRNA agent further comprises a targeting ligand, e.g., a ligand that targets a CNS tissue or a liver tissue. In some embodiments, the CNS tissue is a brain tissue or a spinal tissue, e.g., a dorsal root ganglion.

In some embodiments, the ligand is conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3′ end or the 5′ end of the sense strand. In some embodiments, the ligand is conjugated to the 3′ end of the sense strand.

In some embodiments, the ligand comprises N-acetylgalactosamine (GalNAc). In some embodiments, the targeting ligand comprises one or more GalNAc conjugates or one or more GalNAc derivatives. In some embodiments, the ligand is one or more GalNAc conjugates or one or more GalNAc derivatives are attached through a monovalent linker, or a bivalent, trivalent, or tetravalent branched linker. In some embodiments, the ligand is

In some embodiments, the dsRNA agent is conjugated to the ligand as shown in the following schematic

wherein X is O or S. In some embodiments, the X is O.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first internucleotide linkage at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, second and third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In some embodiments, the dsRNA agent further comprises a phosphate or phosphate mimic at the 5′-end of the antisense strand. In some embodiments, the phosphate mimic is a 5′-vinyl phosphonate (VP).

In some embodiments, a cell described herein, e.g., a human cell, was produced by a process comprising contacting a human cell with the dsRNA agent described herein.

In some embodiments, a pharmaceutical composition described herein comprises the dsRNA agent and a lipid formulation.

In some embodiments (e.g., embodiments of the methods described herein), the cell is within a subject. In some embodiments, the subject is a human. In some embodiments, the level of SCN9A mRNA is inhibited by at least 50%. In some embodiments, the level of SCN9A protein is inhibited by at least 50%. In some embodiments, the expression of SCN9A is inhibited by at least 50%. In some embodiments, inhibiting expression of SCN9A decreases the SCN9A protein level in a biological sample (e.g., a cerebral spinal fluid (CSF) sample, or a CNS biopsy sample) from the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, inhibiting expression of SCN9A gene decreases the SCN9A mRNA level in a biological sample (e.g., a cerebral spinal fluid (CSF) sample, or a CNS biopsy sample) from the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

In some embodiments, the subject has or has been diagnosed with having a SCN9A-associated disorder. In some embodiments, the subject meets at least one diagnostic criterion for a SCN9A-associated disorder. In some embodiments, the SCN9A associated disorder is pain, e.g., chronic pain e.g., inflammatory pain, neuropathic pain, pain hypersensitivity, pain hyposensitivity, inability to sense pain, primary erythromelalgia (PE), paroxysmal extreme pain disorder (PEPD), small fiber neuropathy (SFN), trigeminal neuralgia (TN), and pain associated with, e.g., cancer, arthritis, diabetes, traumatic injury and viral infections.

In some embodiments, the neuronal cell or tissue is a peripheral sensory neuron, e.g., a peripheral sensory neuron in a dorsal root ganglion, or a nociceptive neuron, e.g., an A-delta fiber or a C-type fiber.

In some embodiments, the SCN9A-associated disorder is pain, e.g., chronic pain. In some embodiments, the chronic pain is caused by or associated with pain hypersensitivity, pain hyposensitivity, inability to sense pain, primary erythromelalgia (PE), paroxysmal extreme pain disorder (PEPD), small fiber neuropathy (SFN), trigeminal neuralgia (TN), and pain associated with, e.g., cancer, arthritis, diabetes, traumatic injury or viral infections

In some embodiments, treating comprises amelioration of at least one sign or symptom of the disorder. In some embodiments, the at least one sign or symptom includes a measure of one or more of pain sensitivity, pain threshold, pain level, pain disability level presence, level, or activity of SCN9A (e.g., SCN9A gene, SCN9A mRNA, or SCN9A protein).

In some embodiments, a level of the SCN9A that is higher than a reference level is indicative that the subject has pain, e.g., chronic pain or a pain-related disorder. In some embodiments, treating comprises prevention of progression of the disorder. In some embodiments, the treating comprises one or more of (a) reducing pain; or (b) inhibiting or reducing the expression or activity of SCN9A.

In some embodiments, the treating results in at least a 30% mean reduction from baseline of SCN9A mRNA in the dorsal root ganglion. In some embodiments, the treating results in at least a 60% mean reduction from baseline of SCN9A mRNA in the dorsal root ganglion. In some embodiments, the treating results in at least a 90% mean reduction from baseline of SCN9A mRNA in the dorsal root ganglion.

In some embodiments, after treatment the subject experiences at least an 8-week duration of knockdown following a single dose of dsRNA as assessed by SCN9A protein in the cerebral spinal fluid (CSF) or the CNS tissue, e.g., the dorsal root ganglion. In some embodiments, treating results in at least a 12-week duration of knockdown following a single dose of dsRNA as assessed by SCN9A protein in the cerebral spinal fluid (CSF) or the CNS tissue, e.g., the dorsal root ganglion. In some embodiments, treating results in at least a 16-week duration of knockdown following a single dose of dsRNA as assessed by SCN9A protein in the cerebral spinal fluid (CSF) or the CNS tissue, e.g., the dorsal root ganglion.

In some embodiments, the subject is human.

In some embodiments, the dsRNA agent is administered at a dose of about 0.01 mg/kg to about 50 mg/kg.

In some embodiments, the dsRNA agent is administered to the subject intracranially or intrathecally.

In some embodiments, the dsRNA agent is administered to the subject intrathecally, intraventricularly, or intracerebrally.

In some embodiments, a method described herein further comprises measuring a level of SCN9A (e.g., SCN9A gene, SCN9A mRNA, or SCN9A protein) in the subject. In some embodiments, measuring the level of SCN9A in the subject comprises measuring the level of SCN9A protein in a biological sample from the subject (e.g., a cerebral spinal fluid (CSF) sample or a CNS biopsy sample). In some embodiments, a method described herein further comprises performing a blood test, an imaging test, or, a CNS biopsy, or an aqueous cerebral spinal fluid biopsy.

In some embodiments, a method described herein further measuring level of SCN9A (e.g., SCN9A gene, SCN9A mRNA, or SCN9A protein) in the subject is performed prior to treatment with the dsRNA agent or the pharmaceutical composition. In some embodiments, upon determination that a subject has a level of SCN9A that is greater than a reference level, the dsRNA agent or the pharmaceutical composition is administered to the subject. In some embodiments, measuring level of SCN9A in the subject is performed after treatment with the dsRNA agent or the pharmaceutical composition.

In some embodiments, a method described herein further comprises treating the subject with a therapy suitable for treatment or prevention of a SCN9A-associated disorder, e.g., wherein the therapy comprises non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen, opioids, or corticosteroids, acupuncture, therapeutic massage, dorsal root ganglion stimulation, spinal cord stimulation, or topical pain relievers. In some embodiments, a method described herein further comprises administering to the subject an additional agent suitable for treatment or prevention of a SCN9A-associated disorder. In some embodiments, the additional agent comprises a steroid, or a non-steroidal anti-inflammatory agent.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

The details of various embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and the drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1A depicts the sequences and chemistry of exemplary SCN9A siRNAs including AD-795305, AD-1251249, AD-1251251, AD-1010663, AD-1251301, and AD-961179. FIG. 1B depicts the sequences and chemistry of exemplary SCN9A siRNAs including AD-1251317, AD-1251318, AD-1251323, AD-1251325, AD-795634, AD-1251363. FIG. 1C depicts the sequences and chemistry of exemplary SCN9A siRNAs including AD-1251364, AD-1251373, AD-1251385, AD-1251391, and AD-795913. For each siRNA, “F” is the “2′-fluoro” modification, OMe is a methoxy group, GNA refers to a glycol nucleic acid, “(A2p)” refers to adenosine 2′-phosphate, “(C2p)” refers to cytosine 2′-phosphate, “(G2p)” refers to guanosine 2′-phosphate, “DNA” refers to a DNA base, 2-C16 refers to the targeting ligand, and PS refers to the phosphorothioate linkage. FIGS. 1A-1C disclose SEQ ID NOS 5996-6029, respectively, in order of appearance.

FIG. 2 is a graph depicting the percent SCN9A message remaining relative to PBS in mice on day 14 post-treatment with the exemplary duplexes indicated on the X-axis (from left to right: PBS, AD-795305 (parent), AD-1251249, AD-1251251, AD-1010663 (parent), AD-1251301, AD-961179 (parent), AD-1251317, AD-1251318, AD-1251323, AD-1251325, AD-795634 (parent), AD-1251363, AD-1251364, AD-1251373, AD-1251385, and AD-1251391).

FIG. 3A depicts the sequences and chemistry of exemplary SCN9A siRNAs including AD-802471, AD-1251492, AD-961334, AD-1251279, and AD-1251284. FIG. 3B depicts the sequences and chemistry of exemplary SCN9A siRNAs including AD-1251334, AD-1251377, AD-1251398, AD-1251399, AD-961188, and AD-1251274. FIGS. 3A-3B disclose SEQ ID NOS 6030-6051, respectively, in order of appearance. FIG. 3C depicts the sequences and chemistry of exemplary SCN9A siRNAs including AD-796825, AD-1251411, AD-1251419, AD-797564, AD-1251428, and AD-1251434. FIG. 3D depicts the sequences and chemistry of exemplary SCN9A siRNAs including AD-1010661, AD-795366, AD-795634, and AD-795913. For each siRNA, “F” is the “2′-fluoro” modification, OMe is a methoxy group, GNA refers to a glycol nucleic acid, “(A2p)” refers to adenosine 2′-phosphate, “(C2p)” refers to cytosine 2′-phosphate, “(U2p)” refers to uracil 2′-phosphate, “(G2p)” refers to guanosine 2′-phosphate, “DNA” refers to a DNA base, 2-C16 refers to the targeting ligand, and PS refers to the phosphorothioate linkage. FIGS. 3C-3D disclose SEQ ID NOS 6052-6071, respectively, in order of appearance.

FIGS. 4A-4C present a series of graphs depicting the percent SCN9A message remaining versus the starting position in the target mRNA (NM_001365536.1) of the sense strand of the duplex grouped by those tested in screens 1 and 2 (targeting ORF-1, ORF-2, and the 3′ UTR). FIG. 4A depicts the percent SCN9A message remaining with the duplexes tested at a final concentration of 0.1 nM. FIG. 4B depicts the percent SCN9A message remaining with the duplexes tested at a final concentration of 1 nM. FIG. 4C depicts the percent SCN9A message remaining with the duplexes tested at a final concentration of 10 nM. In FIGS. 4A-4C, screen 1 includes the following duplexes: AD-1010663.3, AD-1251301.1, AD-1251249.1, AD-1251251.1, AD-795305.3, AD-1251363.1, AD-1251364.1, AD-1251373.1, AD-795634.4, AD-1251385.1, AD-1251391.1, AD-1251317.1, AD-1251318.1, AD-1251323.1, AD-1251325.1, and AD-961179.3; screen 2 included the following duplexes: AD-1251492.1, AD-1251279.1, AD-961334.3, AD-1251284.1, AD-1251334.1, AD-1251377.1, AD-1251398.1, AD-1251399.1, AD-1251274.2, AD-961188.3, AD-1251411.1, AD-1251419.1, AD-796825.3, AD-1251428.1, AD-797564.4, and AD-1251434.1.

FIG. 5 is a graph depicting the percent SCN9A message remaining relative to PBS in mice on day 14 post-treatment with the exemplary duplexes indicated on the X-axis (from left to right: PBS, AD-1251492.2*, AD-961334.2 (parent), AD-1251279.2, PBS, AD-1251284.2*, AD-1251334.2*, AD-1251377.2*, AD-1251398.2*, AD-1251399.2*, AD-961188.2 (parent), AD-1251274.2, PBS, AD-796825.2 (parent), AD-1251411.2, AD-1251419.2, AD-797564.3 (parent), AD-1251428.2, and AD-1251434.2. The graph is divided into subsections for those duplexes that target the 3′UTR2 (AD-1251492.2*, AD-961334.2 (parent), AD-1251279.2), ORF1 (AD-1251284.2*, AD-1251334.2*, AD-1251377.2*, AD-1251398.2*, AD-1251399.2*, AD-961188.2 (parent), AD-1251274.2), and ORF2 (AD-796825.2 (parent), AD-1251411.2, AD-1251419.2, AD-797564.3 (parent), AD-1251428.2, AD-1251434.2).

FIG. 6A depicts the sequences and chemistry of exemplary SCN9A siRNAs including AD-1251284, AD-961334, and AD-1251325. FIG. 6A discloses SEQ ID NOS 6072-6077, respectively, in order of appearance. FIG. 6B depicts the sequences and CNS chemistry of exemplary SCN9A duplexes AD-1331352, AD-1209344, and AD-1331350. FIG. 6B discloses SEQ ID NOS 6078-6083, respectively, in order of appearance.

DETAILED DESCRIPTION

iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Described herein are iRNAs and methods of using them for modulating (e.g., inhibiting) the expression of SCN9A. Also provided are compositions and methods for treatment of disorders related to SCN9A expression, such as pain, e.g., acute pain or chronic pain (e.g., inflammatory (nociceptive), neuropathic pain, pain hypersensitivity, pain hyposensitivity, inability to sense pain, primary erythromelalgia (PE), paroxysmal extreme pain disorder (PEPD), small fiber neuropathy (SFN), trigeminal neuralgia (TN), and pain associated with, e.g., cancer, arthritis, diabetes, traumatic injury and viral infections).

Human SCN9A is approximately a 226 kDa protein and is a voltage gated sodium channel (Nav1.7 channel) that mediates the voltage-dependent sodium ion permeability of excitable membranes and also plays a role in nociception signaling. These channels are preferentially expressed in peripheral sensory neurons of the dorsal root ganglia, which are involved in the perception of pain. Mutations in the SCN9A gene have been associated with predispositions to pain hyper- or hyposensitivity. For example, gain-of-function mutations in the SCN9A gene can be the etiological basis of inherited pain syndromes such as primary erythermalgia (PE) and paroxysmal extreme pain disorder (PEPD). Moreover, loss-of-function mutations of the SCN9A gene result in a complete inability of an otherwise healthy individual to sense any form of pain. Without wishing to be bound by theory, increased levels of the SCN9A expression could enhance pain sensitivity; whereas decreased levels of the SCN9A expression could reduce pain sensitivity, and modulating SCN9A expression and Nav1.7 channel levels in peripheral sensory neurons of the dorsal root ganglia could provide an effective pain treatment.

The following description discloses how to make and use compositions containing iRNAs to modulate (e.g., inhibit) the expression of SCN9A, as well as compositions and methods for treating disorders related to expression of SCN9A.

In some aspects, pharmaceutical compositions containing SCN9A iRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of SCN9A, and methods of using the pharmaceutical compositions to treat disorders related to expression of SCN9A (e.g., pain, e.g., chronic pain and/or pain related disorders) are featured herein.

I. Definitions

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary from, for example, between 1% and 15% of the stated number or numerical range.

The terms “or more” and “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 17 nucleotides of a 20-nucleotide nucleic acid molecule” means that 17, 18, 19, or 20 nucleotides have the indicated property. When “at least” is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

As used herein, “or less” and “no more than” are understood as including the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with mismatches to a target site of “no more than 2 nucleotides” has a 2, 1, or 0 mismatches. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.

As used herein, “less than” is understood as not including the value adjacent to the phrase and including logical lower values or integers, as logical from context, to zero. For example, a duplex with mismatches to a target site of “less than 3 nucleotides” has 2, 1, or 0 mismatches. When “less than” is present before a series of numbers or a range, it is understood that “less than” can modify each of the numbers in the series or range.

As used herein, “more than” is understood as not including the value adjacent to the phrase and including logical higher values or integers, as logical from context, to infinity. For example, a duplex with mismatches to a target site of “more than 3 nucleotides” has 4, 5, 6, or more mismatches. When “more than” is present before a series of numbers or a range, it is understood that “more than” can modify each of the numbers in the series or range.

As used herein, “up to” as in “up to 10” is understood as up to and including 10, i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Ranges provided herein are understood to include all individual integer values and all subranges within the ranges.

The terms “activate,” “enhance,” “up-regulate the expression of,” “increase the expression of,” and the like, in so far as they refer to a SCN9A gene, herein refer to the at least partial activation of the expression of a SCN9A gene, as manifested by an increase in the amount of SCN9A mRNA, which may be isolated from or detected in a first cell or group of cells in which a SCN9A gene is transcribed and which has or have been treated such that the expression of a SCN9A gene is increased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).

In some embodiments, expression of a SCN9A gene is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA as described herein. In some embodiments, a SCN9A gene is activated by at least about 60%, 70%, or 80% by administration of an iRNA featured in the disclosure. In some embodiments, expression of a SCN9A gene is activated by at least about 85%, 90%, or 95% or more by administration of an iRNA as described herein. In some embodiments, the SCN9A gene expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold or more in cells treated with an iRNA as described herein compared to the expression in an untreated cell. Activation of expression by small dsRNAs is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103:17337-42, and in US2007/0111963 and US2005/226848, each of which is incorporated herein by reference.

The terms “silence,” “inhibit expression of,” “down-regulate expression of,” “suppress expression of,” and the like, in so far as they refer to SCN9A, herein refer to the at least partial suppression of the expression of SCN9A, as assessed, e.g., based on SCN9A mRNA expression, SCN9A protein expression, or another parameter functionally linked to SCN9A expression. For example, inhibition of SCN9A expression may be manifested by a reduction of the amount of SCN9A mRNA which may be isolated from or detected in a first cell or group of cells in which SCN9A is transcribed and which has or have been treated such that the expression of SCN9A is inhibited, as compared to a control. The control may be a second cell or group of cells substantially identical to the first cell or group of cells, except that the second cell or group of cells have not been so treated (control cells). The degree of inhibition is usually expressed as a percentage of a control level, e.g.,

${\frac{\left( {{mRNA}{in}{control}{cells}} \right) - \left( {{mRNA}{in}{treated}{cells}} \right)}{\left( {{mRNA}{in}{control}{cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to SCN9A expression, e.g., the amount of protein encoded by a SCN9A gene. The reduction of a parameter functionally linked to SCN9A expression may similarly be expressed as a percentage of a control level. In principle, SCN9A silencing may be determined in any cell expressing SCN9A, either constitutively or by genomic engineering, and by any appropriate assay.

For example, in certain instances, expression of SCN9A is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA disclosed herein. In some embodiments, SCN9A is suppressed by at least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA disclosed herein. In some embodiments, SCN9A is suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by administration of an iRNA as described herein.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence.

As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. In some embodiments, the region of complementarity comprises 0, 1, or 2 mismatches.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can be, for example, “stringent conditions”, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.

Complementary sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogsteen base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between two oligonucleotides or polynucleotides, such as the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a SCN9A protein). For example, a polynucleotide is complementary to at least a part of a SCN9A mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding SCN9A. The term “complementarity” refers to the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

As used herein, the term “region of complementarity” refers to the region of one nucleotide sequence agent that is substantially complementary to another sequence, e.g., the region of a sense sequence and corresponding antisense sequence of a dsRNA, or the antisense strand of an iRNA and a target sequence, e.g., a SCN9A nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the antisense strand of the iRNA. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- or 3′-terminus of the iRNA agent.

“Contacting,” as used herein, includes directly contacting a cell, as well as indirectly contacting a cell. For example, a cell within a subject may be contacted when a composition comprising an iRNA is administered (e.g., intrathecally, intracranially, intracerebrally, or intraventricularly) to the subject.

“Introducing into a cell,” when referring to an iRNA, means facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an iRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a β-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or known in the art.

As used herein, a “disorder related to SCN9A expression,” a “disease related to SCN9A expression,” a “pathological process related to SCN9A expression,” “a SCN9A-associated disorder,” “a SCN9A-associated disease,” or the like includes any condition, disorder, or disease in which SCN9A expression is altered (e.g., decreased or increased relative to a reference level, e.g., a level characteristic of a non-diseased subject). In some embodiments, SCN9A expression is decreased. In some embodiments, SCN9A expression is increased. In some embodiments, the decrease or increase in SCN9A expression is detectable in a tissue sample from the subject (e.g., in a cerebral spinal fluid (CSF) sample or a CNS biopsy sample). The decrease or increase may be assessed relative the level observed in the same individual prior to the development of the disorder or relative to other individual(s) who do not have the disorder. The decrease or increase may be limited to a particular organ, tissue, or region of the body (e.g., the brain or the spine). SCN9A-A-associated disorders include, but are not limited to, pain, e.g., chronic pain or pain-related disorders.

“Pain” as defined herein includes acute pain and chronic pain. Chronic pain includes inflammatory (nociceptive) and neuropathic pain associated with disorders including, but not limited to, cancer, arthritis, diabetes, traumatic injury and viral infections. Also included is pain due to inherited pain syndromes including, but not limited to primary erythermalgia (PE) and paroxysmal extreme pain disorder (PEPD).

The term “double-stranded RNA,” “dsRNA,” or “siRNA” as used herein, refers to an iRNA that includes an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA. The duplex region can be of any length that permits specific degradation of a desired target RNA, e.g., through a RISC pathway, but will typically range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length. One strand of the duplex region of a dsDNA comprises a sequence that is substantially complementary to a region of a target RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules. Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop”) between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. In some embodiments, the two strands are connected covalently by means other than a hairpin loop, and the connecting structure is a linker.

In some embodiments, the iRNA agent may be a “single-stranded siRNA” that is introduced into a cell or organism to inhibit a target mRNA. In some embodiments, single-stranded RNAi agents can bind to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are optionally chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein (e.g., sequences provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20) may be used as a single-stranded siRNA as described herein and optionally as chemically modified, e.g., as described herein, e.g., by the methods described in Lima et al., (2012) Cell 150:883-894.

In some embodiments, an RNA interference agent includes a single stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in some embodiments, the disclosure relates to a single stranded RNA that promotes the formation of a RISC complex to effect silencing of the target gene.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the terms “deoxyribonucleotide,” “ribonucleotide,” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the disclosure by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the disclosure.

As used herein, the term “iRNA,” “RNAi”, “iRNA agent,” or “RNAi agent” or “RNAi molecule” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript, e.g., via an RNA-induced silencing complex (RISC) pathway. In some embodiments, an iRNA as described herein effects inhibition of SCN9A expression, e.g., in a cell or mammal. Inhibition of SCN9A expression may be assessed based on a reduction in the level of SCN9A mRNA or a reduction in the level of the SCN9A protein.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound.

The term “lipophile” or “lipophilic moiety” broadly refers to any compound or chemical moiety having an affinity for lipids. One way to characterize the lipophilicity of the lipophilic moiety is by the octanol-water partition coefficient, logK_(ow), where K_(ow) is the ratio of a chemical's concentration in the octanol-phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it may also be predicted by using coefficients attributed to the structural components of a chemical which are calculated using first-principle or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of the tendency of the substance to prefer a non-aqueous or oily milieu rather than water (i.e. its hydrophilic/lipophilic balance). In principle, a chemical substance is lipophilic in character when its logK_(ow) exceeds 0. Typically, the lipophilic moiety possesses a logK_(ow) exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. For instance, the logK_(ow) of 6-amino hexanol, for instance, is predicted to be approximately 0.7. Using the same method, the logK_(ow) of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7.

The lipophilicity of a molecule can change with respect to the functional group it carries. For instance, adding a hydroxyl group or amine group to the end of a lipophilic moiety can increase or decrease the partition coefficient (e.g., logK_(ow)) value of the lipophilic moiety.

Alternatively, the hydrophobicity of the double-stranded RNAi agent, conjugated to one or more lipophilic moieties, can be measured by its protein binding characteristics. For instance, in certain embodiments, the unbound fraction in the plasma protein binding assay of the double-stranded RNAi agent could be determined to positively correlate to the relative hydrophobicity of the double-stranded RNAi agent, which could then positively correlate to the silencing activity of the double-stranded RNAi agent.

In some embodiments, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol of this binding assay is illustrated in detail in, e.g., PCT/US2019/031170. The hydrophobicity of the double-stranded RNAi agent, measured by fraction of unbound siRNA in the binding assay, exceeds 0.15, exceeds 0.2, exceeds 0.25, exceeds 0.3, exceeds 0.35, exceeds 0.4, exceeds 0.45, or exceeds 0.5 for an enhanced in vivo delivery of siRNA.

Accordingly, conjugating the lipophilic moieties to the internal position(s) of the double-stranded RNAi agent provides optimal hydrophobicity for the enhanced in vivo delivery of siRNA.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., a RNAi agent or a plasmid from which a RNAi agent is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

As used herein, the term “modulate the expression of,” refers to an at least partial “inhibition” or partial “activation” of a gene (e.g., SCN9A gene) expression in a cell treated with an iRNA composition as described herein compared to the expression of the corresponding gene in a control cell. A control cell includes an untreated cell, or a cell treated with a non-targeting control iRNA.

The skilled artisan will recognize that the term “RNA molecule” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a “ribonucleoside” includes a nucleoside base and a ribose sugar, and a “ribonucleotide” is a ribonucleoside with one, two or three phosphate moieties or analogs thereof (e.g., phosphorothioate). However, the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure, in the ribose structure, or in the ribose-phosphate backbone structure, e.g., as described herein below. However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2′-O-methyl modified nucleoside, a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, an acyclic nucleoside, a glycol nucleotide, a 2′-deoxy-2′-fluoro modified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, or in combination, an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In some embodiments, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA, e.g., via a RISC pathway. For clarity, it is understood that the term “iRNA” does not encompass a naturally occurring double stranded DNA molecule or a 100% deoxynucleoside-containing DNA molecule.

In some aspects, a modified ribonucleoside includes a deoxyribonucleoside. In such an instance, an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA. In certain embodiments, the RNA molecule comprises a percentage of deoxyribonucleosides of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or higher (but not 100%) deoxyribonucleosides, e.g., in one or both strands.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′ end, 3′ end or both ends of either an antisense or sense strand of a dsRNA.

In some embodiments, the antisense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In some embodiments, the sense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In some embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a therapeutic agent (e.g., an iRNA) and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an agent (e.g., iRNA) effective to produce the intended pharmacological, therapeutic or preventive result. For example, in a method of treating a disorder related to SCN9A expression (e.g., pain, e.g., chronic pain or pain-related disorder), an effective amount includes an amount effective to reduce one or more symptoms associated with the disorder (e.g., an amount effective to (a) inhibit pain or (b) inhibit or reduces the expression or activity of SCN9A) or an amount effective to reduce the risk of developing conditions associated with the disorder. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to obtain at least a 10% reduction in that parameter. For example, a therapeutically effective amount of an iRNA targeting SCN9A can reduce a level of SCN9A mRNA or a level of SCN9A protein by any measurable amount, e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in drug formulations are described further herein below.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 2006/0240093, 2007/0135372, and in International Application No. WO 2009/082817. These applications are incorporated herein by reference in their entirety. In some embodiments, the SNALP is a SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

As used herein, a “subject” to be treated according to the methods described herein, includes a human or non-human animal, e.g., a mammal. The mammal may be, for example, a rodent (e.g., a rat or mouse) or a primate (e.g., a monkey). In some embodiments, the subject is a human.

A “subject in need thereof” includes a subject having, suspected of having, or at risk of developing a disorder related to SCN9A expression, e.g., overexpression (e.g., pain, e.g., chronic pain or a pain-related disorder). In some embodiments, the subject has, or is suspected of having, a disorder related to SCN9A expression or overexpression. In some embodiments, the subject is at risk of developing a disorder related to SCN9A expression or overexpression.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene, e.g., SCN9A, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” and the like refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of any disorder or pathological process related to SCN9A expression (e.g., pain, e.g., chronic pain or a pain-related disorder). The specific amount that is therapeutically effective may vary depending on factors known in the art, such as, for example, the type of disorder or pathological process, the patient's history and age, the stage of the disorder or pathological process, and the administration of other therapies.

In the context of the present disclosure, the terms “treat,” “treatment,” and the like mean to prevent, delay, relieve or alleviate at least one symptom associated with a disorder related to SCN9A expression, or to slow or reverse the progression or anticipated progression of such a disorder. For example, the methods featured herein, when employed to treat pain, e.g., chronic pain or a pain-related disorder, may serve to reduce or prevent one or more symptoms of the pain, e.g., chronic pain, as described herein, or to reduce the risk or severity of associated conditions. Thus, unless the context clearly indicates otherwise, the terms “treat,” “treatment,” and the like are intended to encompass prophylaxis, e.g., prevention of disorders and/or symptoms of disorders related to SCN9A expression. Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.

By “lower” in the context of a disease marker or symptom is meant any decrease, e.g., a statistically or clinically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. The decrease can be down to a level accepted as within the range of normal for an individual without such disorder.

As used herein, “SCN9A” refers to “sodium channel, voltage gated, type IX alpha subunit” gene (“SCN9A gene”), the corresponding mRNA (“SCN9A mRNA”), or the corresponding protein (“SCN9A protein”). The sequence of a human SCN9A mRNA transcript can be found at SEQ ID NO: 1 or SEQ ID NO: 4001.

In the event of a discrepancy between the recited positions of the duplexes presented herein and the alignment of the duplexes to the recited sequences, the alignment of the duplexes to the recited sequence will govern.

II. iRNA Agents

Described herein are iRNA agents that modulate (e.g., inhibit) the expression of SCN9A.

In some embodiments, the iRNA agent activates the expression of SCN9A in a cell or mammal.

In some embodiments, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of SCN9A in a cell or in a subject (e.g., in a mammal, e.g., in a human), where the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of SCN9A, and where the region of complementarity is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and where the dsRNA, upon contact with a cell expressing SCN9A, inhibits the expression of SCN9A, e.g., by at least 10%, 20%, 30%, 40%, or 50%.

The modulation (e.g., inhibition) of expression of SCN9A can be assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. Expression of SCN9A in cell culture, such as in COS cells, ARPE-19 cells, hTERT RPE-1 cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured cells or in a biological sample from a subject can be assayed by measuring SCN9A mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by immunofluorescence analysis, using, for example, Western Blotting or flow cytometric techniques.

A dsRNA typically includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) typically includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of SCN9A. The other strand (the sense strand) typically includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive.

In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, e.g., 15-30 nucleotides in length.

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of 9 to 36, e.g., 15-30 base pairs. Thus, in some embodiments, to the extent that it becomes processed to a functional duplex of e.g., 15-30 base pairs that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in some embodiments, then, an miRNA is a dsRNA. In some embodiments, a dsRNA is not a naturally occurring miRNA. In some embodiments, an iRNA agent useful to target SCN9A expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein may further include one or more single-stranded nucleotide overhangs. The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

In some embodiments, SCN9A is a human SCN9A.

In specific embodiments, the dsRNA comprises a sense strand that comprises or consists of a sense sequence selected from the sense sequences provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20 and an antisense strand that comprises or consists of an antisense sequence selected from the antisense sequences provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20.

In some aspects, a dsRNA will include at least sense and antisense nucleotide sequences, whereby the sense strand is selected from the sequences provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20 and the corresponding antisense strand is selected from the sequences provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20.

In these aspects, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated by the expression of SCN9A. As such, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand, and the second oligonucleotide is described as the corresponding antisense strand. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can be effective as well.

In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20, dsRNAs described herein can include at least one strand of a length of minimally 19 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 minus only a few nucleotides on one or both ends will be similarly effective as compared to the dsRNAs described above.

In some embodiments, the dsRNA has a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20.

In some embodiments, the dsRNA has an antisense sequence that comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides of an antisense sequence provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20 and a sense sequence that comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides of a corresponding sense sequence provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20.

In some embodiments, the dsRNA comprises an antisense sequence that comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of an antisense sequence provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20 and a sense sequence that comprises at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of a corresponding sense sequence provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20.

In some such embodiments, the dsRNA, although it comprises only a portion of the sequences provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20 is equally effective in inhibiting a level of SCN9A expression as is a dsRNA that comprises the full-length sequences provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20. In some embodiments, the dsRNA differs in its inhibition of a level of expression of SCN9A by not more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% inhibition compared with a dsRNA comprising the full sequence disclosed herein.

In some embodiments, an iRNA of Table 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20 decreases SCN9A protein or SCN9A mRNA levels in a cell. In some embodiments, the cell is a rodent cell (e.g., a rat cell), or a primate cell (e.g., a cynomolgus monkey cell or a human cell). In some embodiments, SCN9A protein or SCN9A mRNA levels are reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the iRNA of Table 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, or 20 that inhibits SCN9A in a human cell has less than 5, 4, 3, 2, or 1 mismatches to the corresponding portion of human SCN9A. In some embodiments, the iRNA of Table 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 that inhibits SCN9A in a human cell has no mismatches to the corresponding portion of human SCN9A.

iRNAs designed based on human sequences can have utility, e.g., for inhibiting SCN9A in human cells, e.g., for therapeutic purposes, or for inhibiting SCN9A in rodent cells, e.g., for research characterizing SCN9A in a rodent model.

In some embodiments, an iRNA described herein comprises an antisense strand comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2. In some embodiments, an iRNA described herein comprises a sense strand comprising at least 15 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

A human SCN9A mRNA may have the sequence of SEQ ID NO: 1 provided herein.

Homo sapiens Sodium Channel, Voltage Gated, Type IX Alpha Subunit (SCN9A), Transcript Variant 1, mRNA

(SEQ ID NO: 1) CGGGGCUGCUACCUCCACGGGCGCGCCCUGGCAGGAGGGGCGCAGUCUGCUUGCAGGCGGUCGCCAGCGC UCCAGCGGCGGCUGUCGGCUUUCCAAUUCCGCCAGCUCGGCUGAGGCUGGGCUAGCCUGGGUGCCAGUGG CUGCUAGCGGCAGGCGUCCCCUGAGCAACAGGAGCCCAGAGAAAAAGAAGCAGCCCUGAGAGAGCGCCGG GGAAGGAGAGGCCCGCGCCCUCUCCUGGAGCCAGAUUCUGCAGGUGCACUGGGUGGGGAUGAUCGGCGGG CUAGGUUGCAAGCCUCUUAUGUGAGGAGCUGAAGAGGAAUUAAAAUAUACAGGAUGAAAAGAUGGCAAUG UUGCCUCCCCCAGGACCUCAGAGCUUUGUCCAUUUCACAAAACAGUCUCUUGCCCUCAUUGAACAACGCA UUGCUGAAAGAAAAUCAAAGGAACCCAAAGAAGAAAAGAAAGAUGAUGAUGAAGAAGCCCCAAAGCCAAG CAGUGACUUGGAAGCUGGCAAACAGCUGCCCUUCAUCUAUGGGGACAUUCCUCCCGGCAUGGUGUCAGAG CCCCUGGAGGACUUGGACCCCUACUAUGCAGACAAAAAGACUUUCAUAGUAUUGAACAAAGGGAAAACAA UCUUCCGUUUCAAUGCCACACCUGCUUUAUAUAUGCUUUCUCCUUUCAGUCCUCUAAGAAGAAUAUCUAU UAAGAUUUUAGUACACUCCUUAUUCAGCAUGCUCAUCAUGUGCACUAUUCUGACAAACUGCAUAUUUAUG ACCAUGAAUAACCCACCGGACUGGACCAAAAAUGUCGAGUACACUUUUACUGGAAUAUAUACUUUUGAAU CACUUGUAAAAAUCCUUGCAAGAGGCUUCUGUGUAGGAGAAUUCACUUUUCUUCGUGACCCGUGGAACUG GCUGGAUUUUGUCGUCAUUGUUUUUGCGUAUUUAACAGAAUUUGUAAACCUAGGCAAUGUUUCAGCUCUU CGAACUUUCAGAGUAUUGAGAGCUUUGAAAACUAUUUCUGUAAUCCCAGGCCUGAAGACAAUUGUAGGGG CUUUGAUCCAGUCAGUGAAGAAGCUUUCUGAUGUCAUGAUCCUGACUGUGUUCUGUCUGAGUGUGUUUGC ACUAAUUGGACUACAGCUGUUCAUGGGAAACCUGAAGCAUAAAUGUUUUCGAAAUUCACUUGAAAAUAAU GAAACAUUAGAAAGCAUAAUGAAUACCCUAGAGAGUGAAGAAGACUUUAGAAAAUAUUUUUAUUACUUGG AAGGAUCCAAAGAUGCUCUCCUUUGUGGUUUCAGCACAGAUUCAGGUCAGUGUCCAGAGGGGUACACCUG UGUGAAAAUUGGCAGAAACCCUGAUUAUGGCUACACGAGCUUUGACACUUUCAGCUGGGCCUUCUUAGCC UUGUUUAGGCUAAUGACCCAAGAUUACUGGGAAAACCUUUACCAACAGACGCUGCGUGCUGCUGGCAAAA CCUACAUGAUCUUCUUUGUCGUAGUGAUUUUCCUGGGCUCCUUUUAUCUAAUAAACUUGAUCCUGGCUGU GGUUGCCAUGGCAUAUGAAGAACAGAACCAGGCAAACAUUGAAGAAGCUAAACAGAAAGAAUUAGAAUUU CAACAGAUGUUAGACCGUCUUAAAAAAGAGCAAGAAGAAGCUGAGGCAAUUGCAGCGGCAGCGGCUGAAU AUACAAGUAUUAGGAGAAGCAGAAUUAUGGGCCUCUCAGAGAGUUCUUCUGAAACAUCCAAACUGAGCUC UAAAAGUGCUAAAGAAAGAAGAAACAGAAGAAAGAAAAAGAAUCAAAAGAAGCUCUCCAGUGGAGAGGAA AAGGGAGAUGCUGAGAAAUUGUCGAAAUCAGAAUCAGAGGACAGCAUCAGAAGAAAAAGUUUCCACCUUG GUGUCGAAGGGCAUAGGCGAGCACAUGAAAAGAGGUUGUCUACCCCCAAUCAGUCACCACUCAGCAUUCG UGGCUCCUUGUUUUCUGCAAGGCGAAGCAGCAGAACAAGUCUUUUUAGUUUCAAAGGCAGAGGAAGAGAU AUAGGAUCUGAGACUGAAUUUGCCGAUGAUGAGCACAGCAUUUUUGGAGACAAUGAGAGCAGAAGGGGCU CACUGUUUGUGCCCCACAGACCCCAGGAGCGACGCAGCAGUAACAUCAGCCAAGCCAGUAGGUCCCCACC AAUGCUGCCGGUGAACGGGAAAAUGCACAGUGCUGUGGACUGCAACGGUGUGGUCUCCCUGGUUGAUGGA CGCUCAGCCCUCAUGCUCCCCAAUGGACAGCUUCUGCCAGAGGGCACGACCAAUCAAAUACACAAGAAAA GGCGUUGUAGUUCCUAUCUCCUUUCAGAGGAUAUGCUGAAUGAUCCCAACCUCAGACAGAGAGCAAUGAG UAGAGCAAGCAUAUUAACAAACACUGUGGAAGAACUUGAAGAGUCCAGACAAAAAUGUCCACCUUGGUGG UACAGAUUUGCACACAAAUUCUUGAUCUGGAAUUGCUCUCCAUAUUGGAUAAAAUUCAAAAAGUGUAUCU AUUUUAUUGUAAUGGAUCCUUUUGUAGAUCUUGCAAUUACCAUUUGCAUAGUUUUAAACACAUUAUUUAU GGCUAUGGAACACCACCCAAUGACUGAGGAAUUCAAAAAUGUACUUGCUAUAGGAAAUUUGGUCUUUACU GGAAUCUUUGCAGCUGAAAUGGUAUUAAAACUGAUUGCCAUGGAUCCAUAUGAGUAUUUCCAAGUAGGCU GGAAUAUUUUUGACAGCCUUAUUGUGACUUUAAGUUUAGUGGAGCUCUUUCUAGCAGAUGUGGAAGGAUU GUCAGUUCUGCGAUCAUUCAGACUGCUCCGAGUCUUCAAGUUGGCAAAAUCCUGGCCAACAUUGAACAUG CUGAUUAAGAUCAUUGGUAACUCAGUAGGGGCUCUAGGUAACCUCACCUUAGUGUUGGCCAUCAUCGUCU UCAUUUUUGCUGUGGUCGGCAUGCAGCUCUUUGGUAAGAGCUACAAAGAAUGUGUCUGCAAGAUCAAUGA UGACUGUACGCUCCCACGGUGGCACAUGAACGACUUCUUCCACUCCUUCCUGAUUGUGUUCCGCGUGCUG UGUGGAGAGUGGAUAGAGACCAUGUGGGACUGUAUGGAGGUCGCUGGUCAAGCUAUGUGCCUUAUUGUUU ACAUGAUGGUCAUGGUCAUUGGAAACCUGGUGGUCCUAAACCUAUUUCUGGCCUUAUUAUUGAGCUCAUU UAGUUCAGACAAUCUUACAGCAAUUGAAGAAGACCCUGAUGCAAACAACCUCCAGAUUGCAGUGACUAGA AUUAAAAAGGGAAUAAAUUAUGUGAAACAAACCUUACGUGAAUUUAUUCUAAAAGCAUUUUCCAAAAAGC CAAAGAUUUCCAGGGAGAUAAGACAAGCAGAAGAUCUGAAUACUAAGAAGGAAAACUAUAUUUCUAACCA UACACUUGCUGAAAUGAGCAAAGGUCACAAUUUCCUCAAGGAAAAAGAUAAAAUCAGUGGUUUUGGAAGC AGCGUGGACAAACACUUGAUGGAAGACAGUGAUGGUCAAUCAUUUAUUCACAAUCCCAGCCUCACAGUGA CAGUGCCAAUUGCACCUGGGGAAUCCGAUUUGGAAAAUAUGAAUGCUGAGGAACUUAGCAGUGAUUCGGA UAGUGAAUACAGCAAAGUGAGAUUAAACCGGUCAAGCUCCUCAGAGUGCAGCACAGUUGAUAACCCUUUG CCUGGAGAAGGAGAAGAAGCAGAGGCUGAACCUAUGAAUUCCGAUGAGCCAGAGGCCUGUUUCACAGAUG GUUGUGUACGGAGGUUCUCAUGCUGCCAAGUUAACAUAGAGUCAGGGAAAGGAAAAAUCUGGUGGAACAU CAGGAAAACCUGCUACAAGAUUGUUGAACACAGUUGGUUUGAAAGCUUCAUUGUCCUCAUGAUCCUGCUC AGCAGUGGUGCCCUGGCUUUUGAAGAUAUUUAUAUUGAAAGGAAAAAGACCAUUAAGAUUAUCCUGGAGU AUGCAGACAAGAUCUUCACUUACAUCUUCAUUCUGGAAAUGCUUCUAAAAUGGAUAGCAUAUGGUUAUAA AACAUAUUUCACCAAUGCCUGGUGUUGGCUGGAUUUCCUAAUUGUUGAUGUUUCUUUGGUUACUUUAGUG GCAAACACUCUUGGCUACUCAGAUCUUGGCCCCAUUAAAUCCCUUCGGACACUGAGAGCUUUAAGACCUC UAAGAGCCUUAUCUAGAUUUGAAGGAAUGAGGGUCGUUGUGAAUGCACUCAUAGGAGCAAUUCCUUCCAU CAUGAAUGUGCUACUUGUGUGUCUUAUAUUCUGGCUGAUAUUCAGCAUCAUGGGAGUAAAUUUGUUUGCU GGCAAGUUCUAUGAGUGUAUUAACACCACAGAUGGGUCACGGUUUCCUGCAAGUCAAGUUCCAAAUCGUU CCGAAUGUUUUGCCCUUAUGAAUGUUAGUCAAAAUGUGCGAUGGAAAAACCUGAAAGUGAACUUUGAUAA UGUCGGACUUGGUUACCUAUCUCUGCUUCAAGUUGCAACUUUUAAGGGAUGGACGAUUAUUAUGUAUGCA GCAGUGGAUUCUGUUAAUGUAGACAAGCAGCCCAAAUAUGAAUAUAGCCUCUACAUGUAUAUUUAUUUUG UCGUCUUUAUCAUCUUUGGGUCAUUCUUCACUUUGAACUUGUUCAUUGGUGUCAUCAUAGAUAAUUUCAA CCAACAGAAAAAGAAGCUUGGAGGUCAAGACAUCUUUAUGACAGAAGAACAGAAGAAAUACUAUAAUGCA AUGAAAAAGCUGGGGUCCAAGAAGCCACAAAAGCCAAUUCCUCGACCAGGGAACAAAAUCCAAGGAUGUA UAUUUGACCUAGUGACAAAUCAAGCCUUUGAUAUUAGUAUCAUGGUUCUUAUCUGUCUCAACAUGGUAAC CAUGAUGGUAGAAAAGGAGGGUCAAAGUCAACAUAUGACUGAAGUUUUAUAUUGGAUAAAUGUGGUUUUU AUAAUCCUUUUCACUGGAGAAUGUGUGCUAAAACUGAUCUCCCUCAGACACUACUACUUCACUGUAGGAU GGAAUAUUUUUGAUUUUGUGGUUGUGAUUAUCUCCAUUGUAGGUAUGUUUCUAGCUGAUUUGAUUGAAAC GUAUUUUGUGUCCCCUACCCUGUUCCGAGUGAUCCGUCUUGCCAGGAUUGGCCGAAUCCUACGUCUAGUC AAAGGAGCAAAGGGGAUCCGCACGCUGCUCUUUGCUUUGAUGAUGUCCCUUCCUGCGUUGUUUAACAUCG GCCUCCUGCUCUUCCUGGUCAUGUUCAUCUACGCCAUCUUUGGAAUGUCCAACUUUGCCUAUGUUAAAAA GGAAGAUGGAAUUAAUGACAUGUUCAAUUUUGAGACCUUUGGCAACAGUAUGAUUUGCCUGUUCCAAAUU ACAACCUCUGCUGGCUGGGAUGGAUUGCUAGCACCUAUUCUUAACAGUAAGCCACCCGACUGUGACCCAA AAAAAGUUCAUCCUGGAAGUUCAGUUGAAGGAGACUGUGGUAACCCAUCUGUUGGAAUAUUCUACUUUGU UAGUUAUAUCAUCAUAUCCUUCCUGGUUGUGGUGAACAUGUACAUUGCAGUCAUACUGGAGAAUUUUAGU GUUGCCACUGAAGAAAGUACUGAACCUCUGAGUGAGGAUGACUUUGAGAUGUUCUAUGAGGUUUGGGAGA AGUUUGAUCCCGAUGCGACCCAGUUUAUAGAGUUCUCUAAACUCUCUGAUUUUGCAGCUGCCCUGGAUCC UCCUCUUCUCAUAGCAAAACCCAACAAAGUCCAGCUCAUUGCCAUGGAUCUGCCCAUGGUUAGUGGUGAC CGGAUCCAUUGUCUUGACAUCUUAUUUGCUUUUACAAAGCGUGUUUUGGGUGAGAGUGGGGAGAUGGAUU CUCUUCGUUCACAGAUGGAAGAAAGGUUCAUGUCUGCAAAUCCUUCCAAAGUGUCCUAUGAACCCAUCAC AACCACACUAAAACGGAAACAAGAGGAUGUGUCUGCUACUGUCAUUCAGCGUGCUUAUAGACGUUACCGC UUAAGGCAAAAUGUCAAAAAUAUAUCAAGUAUAUACAUAAAAGAUGGAGACAGAGAUGAUGAUUUACUCA AUAAAAAAGAUAUGGCUUUUGAUAAUGUUAAUGAGAACUCAAGUCCAGAAAAAACAGAUGCCACUUCAUC CACCACCUCUCCACCUUCAUAUGAUAACAAAGCCAGACAAAGAGAAAUAUGAACAAGACAGAACAGAAAA GGAAGACAAAGGGAAAGACAGCAAGGAAAGCAAAAAAUAGAGCUUCAUUUUUGAUAUAUUGUUUACAGCC UGUGAAAGUGAUUUAUUUGUGUUAAUAAAACUCUUUUGAGGAAGUCUAUGCCAAAAUCCUUUUUAUCAAA AUAUUCUCGAAGGCAGUGCAGUCACUAACUCUGAUUUCCUAAGAAAGGUGGGCAGCAUUAGCAGAUGGUU AUUUUUGCACUGAUGAUUCUUUAAGAAUCGUAAGAGAACUCUGUAGGAAUUAUUGAUUAUAGCAUACAAA AGUGAUUCAGUUUUUUGGUUUUUAAUAAAUCAGAAGACCAUGUAGAAAACUUUUACAUCUGCCUUGUCAU CUUUUCACAGGAUUGUAAUUAGUCUUGUUUCCCAUGUAAAUAAACAACACACGCAUACAGAAAAAUCUAU UAUUUAUCUAUUAUUUGGAAAUCAACAAAAGUAUUUGCCUUGGCUUUGCAAUGAAAUGCUUGAUAGAAGU AAUGGACAUUAGUUAUGAAUGUUUAGUUAAAAUGCAUUAUUAGGGAGCUUGACUUUUUAUCAAUGUACAG AGGUUAUUCUAUAUUUUGAGGUGCUUAAAUUUAUUCUACAUUGCAUCAGAACCAAUUUAUAUGUGCCUAU AAAAUGCCAUGGGAUUAAAAAUAUAUGUAGGCUAUUCAUUUCUACAAAUGUUUUUCAUUCAUCUUGACUC ACAUGCCAACAAGGAUAAGACUUACCUUUAGAGUAUUGUGUUUCAUAGCCUUUCUUCUUUCAUAUCCCUU UUUGUUCAUAGAAUAACCACAGAACUUGAAAAAUUAUUCUAAGUACAUAUUACACUCCUCAAAAAAAACA AAGAUAACUGAGAAAAAAGUUAUUGACAGAAGUUCUAUUUGCUAUUAUUUACAUAGCCUAACAUUUGACU GUGCUGCCCAAAAUACUGAUAAUAGUCUCUUAAACUCUUUUGUCAAAUUUUCCUGCUUUCUUAUGCAGUA UUGUUUAGUCAUCCUUUCGCUGUAAGCAAAGUUGAUGAAAUCCUUCCUGAUAUGCAGUUAGUUGUUUGAC CACGGUACAUACUUGAGCAGAUAAUAACUUGGGCACAGUAUUUAUUGCAUCACUUGUAUACAAUCCCGUG UUUGGCAAGCUUUCAAAUCAUGUAAUAUGACAGACUUUACACAGAUAUGUGUUUAGUAUGAAUAAAAAAG CAUUGAAAUAGGGAUUCUUGCCAACUUGCUCUCUUGCCACCAACUUACUUUCCUAAAUUAUGGAAGUAAU CUUUUUUGGAUAUACUUCAAUGUAUACAAUGAGGAAGAUGUCACCUUCUCCUUAAAAUUCUAUGAUGUGA AAUAUAUUUUGCCUCAAUCAACACAGUACCAUGGGCUUCUAAUUUAUCAAGCACAUAUUCAUUUUGCAUU AGCUGUAGACAUCUAGUUUUUUGAAAACACCUAUUAAUAGUAAUUUGAAAAGAAAUAACCAUAAUGCUUU UUUUCGUGAGUUUAUUUCAGGAAUAUGAGAUCUUUCUUCUAUAAAGUUAUUCAUGCACAGGCAAAAAUUG AGCUACACAGGUAGAAUGUAGUUUUACUUAGAAGAUUUUUGUGGGAGGUUUUGAAGCAAAUAUAUAAAAC AACUUUCACUAAUUUGCUUUCCAUAUUUAAAAAAUAAUAAAUUACAUUUAUAUAAUAAAUGUUUAAAGCA CAUAUUUUUUGUUGUUCUGGCAAUUUAAAAAGAAAGAGGAUUUAAACGUACCUAUAGAAACAAAGAUUUA UGGUUAAAGAAUGAGAUCAGAAGUCUAGAAUGUUUUUAAAUUGUGAUAUAUUUUACAACAUCCGUUAUUA CUUUGAGACAUUUGUCCUAAUCUACGUAUAAAACUCAAUCUAGGGCUAAAGAUUCUUUAUACCAUCUUAG GUUCAUUCAUCUUAGGCUAUUUGAACCACUUUUUAAUUUAAUAUGAAAGACACCAUGCAGUGUUUUCCGA GACUACAUAGAUCAUUUUAUCACAUACCUACCAAGCCUGUUGGAAAUAGGUUUUGAUAAUUUAAGUAGGG ACCUAUACAAAAUAUAUUACAUUUAUCAGAUUUUUAAAUACAUUCAAUUAAGAAUUUAACAUCACCUUAA AUUUGAAUUCAAUCUACCGUUAUUUCAAACUCACAAAUAUAACUGCAUUAUGAAUACUUACAUAAUGUAG UAAGACAAGAUGUUUGACAGGUUCGUGUGUAAUUUUCUAUUAAUGUUUUUACAUUGCCUUGUUUUUAUGU AAAAUAAAAAAUAUGGGCAACUGGUUUGUUAACAACACAAUUUCUUCUUAGCAUUUCAAAAAUAUAUAUA AAGUUGUUCUUUUUCCUAUUUCAUGAACUAUGUUUUUUUUUAAAAUAACAUGGUUAAGUUUUAUAUAUAU UUACGUUUGUUUCAGGAAUGUCUACUUGUGACUUUUUAUCAAUUAAAAAUAAUAUUUGGAAGAAAGAGCU UAUUAAGUAUAAGCUUGAAGUAAAAUUAGACCUCUCUUUCCAUGUAGAUUACUGUUUGUACUGAUGGUUU CACCCUUCAGAAGGCACUGUCAUAUUAAUAUUUAAAUUUUAUAAUCGCUGAACUUAUUACACCCAACAAU ACAGAAAGGCAGUUACACUGAAGAACUUAACUUAGAAUAAAAUGGAAGCAAACAGGUUUUCUAAAAACUU UUUUAAGUGACCAGGUCUCGCUCUGUCACCCAGGCUAGAGUGCAAUGGCAUGAUCAUAGCUCUCUGCAGC CUCAACUCUGGGCUCAAGCAACCCUCCUGCCUCAGCCUCCCAAGUAGCUAAGACUACAGGUACAUGCCAC CAUGCCUGGCUAAUAUUUAAAUUUUUGUAGAUAAGGGGUCUUGCUAUGUUGCCCAGGCUAGUCUCAAACU CCUGGCUUCAAGUGUUCCUACUGUCAUGACCUGCCAACAUGCUGGGGUUACAGGCAUGAGCCACCAUGCC CCAAACAGGUUUGAACACAAAUCUUUCGGAUGAAAAUUAGAGAACCUAAUUUUAGCUUUUUGAUAGUUAC CUAGUUUGCAAAAGAUUUGGGUGACUUGUGAGCUGUUUUUAAAUGCUGAUUGUUGAACAUCACAACCCAA AAUACUUAGCAUGAUUUUAUAGAGUUUUGAUAGCUUUAUUAAAAAGAGUGAAAAUAAAAUGCAUAUGUAA AUAAAGCAGUUCUAAAUAGCUAUUUCAGAGAAAUGUUAAUAGAAGUGCUGAAAGAAGGGCCAACUAAAUU AGGAUGGCCAGGGAAUUGGCCUGGGUUUAGGACCUAUGUAUGAAGGCCACCAAUUUUUUAAAAAUAUCUG UGGUUUAUUAUGUUAUUAUCUUCUUGAGGAAAACAAUCAAGAAUUGCUUCAUGAAAAUAAAUAAAUAGCC AUGAAUAUCAUAAAGCUGUUUACAUAGGAUUCUUUACAAAUUUCAUAGAUCUAUGAAUGCUCAAAAUGUU UGAGUUUGCCAUAAAUUAUAUUGUAGUUAUAUUGUAGUUAUACUUGAGACUGACACAUUGUAAUAUAAUC UAAGAAUAAAAGUUAUACAAAAUAAAAAAAAAAAAA

The reverse complement of SEQ ID NO: 1 is provided as SEQ ID NO: 2 herein:

(SEQ ID NO: 2) UUUUUUUUUUUUUAUUUUGUAUAACUUUUAUUCUUAGAUUAUAUUACAAUGUGUCAGUCUCAAGUAUAAC UACAAUAUAACUACAAUAUAAUUUAUGGCAAACUCAAACAUUUUGAGCAUUCAUAGAUCUAUGAAAUUUG UAAAGAAUCCUAUGUAAACAGCUUUAUGAUAUUCAUGGCUAUUUAUUUAUUUUCAUGAAGCAAUUCUUGA UUGUUUUCCUCAAGAAGAUAAUAACAUAAUAAACCACAGAUAUUUUUAAAAAAUUGGUGGCCUUCAUACA UAGGUCCUAAACCCAGGCCAAUUCCCUGGCCAUCCUAAUUUAGUUGGCCCUUCUUUCAGCACUUCUAUUA ACAUUUCUCUGAAAUAGCUAUUUAGAACUGCUUUAUUUACAUAUGCAUUUUAUUUUCACUCUUUUUAAUA AAGCUAUCAAAACUCUAUAAAAUCAUGCUAAGUAUUUUGGGUUGUGAUGUUCAACAAUCAGCAUUUAAAA ACAGCUCACAAGUCACCCAAAUCUUUUGCAAACUAGGUAACUAUCAAAAAGCUAAAAUUAGGUUCUCUAA UUUUCAUCCGAAAGAUUUGUGUUCAAACCUGUUUGGGGCAUGGUGGCUCAUGCCUGUAACCCCAGCAUGU UGGCAGGUCAUGACAGUAGGAACACUUGAAGCCAGGAGUUUGAGACUAGCCUGGGCAACAUAGCAAGACC CCUUAUCUACAAAAAUUUAAAUAUUAGCCAGGCAUGGUGGCAUGUACCUGUAGUCUUAGCUACUUGGGAG GCUGAGGCAGGAGGGUUGCUUGAGCCCAGAGUUGAGGCUGCAGAGAGCUAUGAUCAUGCCAUUGCACUCU AGCCUGGGUGACAGAGCGAGACCUGGUCACUUAAAAAAGUUUUUAGAAAACCUGUUUGCUUCCAUUUUAU UCUAAGUUAAGUUCUUCAGUGUAACUGCCUUUCUGUAUUGUUGGGUGUAAUAAGUUCAGCGAUUAUAAAA UUUAAAUAUUAAUAUGACAGUGCCUUCUGAAGGGUGAAACCAUCAGUACAAACAGUAAUCUACAUGGAAA GAGAGGUCUAAUUUUACUUCAAGCUUAUACUUAAUAAGCUCUUUCUUCCAAAUAUUAUUUUUAAUUGAUA AAAAGUCACAAGUAGACAUUCCUGAAACAAACGUAAAUAUAUAUAAAACUUAACCAUGUUAUUUUAAAAA AAAACAUAGUUCAUGAAAUAGGAAAAAGAACAACUUUAUAUAUAUUUUUGAAAUGCUAAGAAGAAAUUGU GUUGUUAACAAACCAGUUGCCCAUAUUUUUUAUUUUACAUAAAAACAAGGCAAUGUAAAAACAUUAAUAG AAAAUUACACACGAACCUGUCAAACAUCUUGUCUUACUACAUUAUGUAAGUAUUCAUAAUGCAGUUAUAU UUGUGAGUUUGAAAUAACGGUAGAUUGAAUUCAAAUUUAAGGUGAUGUUAAAUUCUUAAUUGAAUGUAUU UAAAAAUCUGAUAAAUGUAAUAUAUUUUGUAUAGGUCCCUACUUAAAUUAUCAAAACCUAUUUCCAACAG GCUUGGUAGGUAUGUGAUAAAAUGAUCUAUGUAGUCUCGGAAAACACUGCAUGGUGUCUUUCAUAUUAAA UUAAAAAGUGGUUCAAAUAGCCUAAGAUGAAUGAACCUAAGAUGGUAUAAAGAAUCUUUAGCCCUAGAUU GAGUUUUAUACGUAGAUUAGGACAAAUGUCUCAAAGUAAUAACGGAUGUUGUAAAAUAUAUCACAAUUUA AAAACAUUCUAGACUUCUGAUCUCAUUCUUUAACCAUAAAUCUUUGUUUCUAUAGGUACGUUUAAAUCCU CUUUCUUUUUAAAUUGCCAGAACAACAAAAAAUAUGUGCUUUAAACAUUUAUUAUAUAAAUGUAAUUUAU UAUUUUUUAAAUAUGGAAAGCAAAUUAGUGAAAGUUGUUUUAUAUAUUUGCUUCAAAACCUCCCACAAAA AUCUUCUAAGUAAAACUACAUUCUACCUGUGUAGCUCAAUUUUUGCCUGUGCAUGAAUAACUUUAUAGAA GAAAGAUCUCAUAUUCCUGAAAUAAACUCACGAAAAAAAGCAUUAUGGUUAUUUCUUUUCAAAUUACUAU UAAUAGGUGUUUUCAAAAAACUAGAUGUCUACAGCUAAUGCAAAAUGAAUAUGUGCUUGAUAAAUUAGAA GCCCAUGGUACUGUGUUGAUUGAGGCAAAAUAUAUUUCACAUCAUAGAAUUUUAAGGAGAAGGUGACAUC UUCCUCAUUGUAUACAUUGAAGUAUAUCCAAAAAAGAUUACUUCCAUAAUUUAGGAAAGUAAGUUGGUGG CAAGAGAGCAAGUUGGCAAGAAUCCCUAUUUCAAUGCUUUUUUAUUCAUACUAAACACAUAUCUGUGUAA AGUCUGUCAUAUUACAUGAUUUGAAAGCUUGCCAAACACGGGAUUGUAUACAAGUGAUGCAAUAAAUACU GUGCCCAAGUUAUUAUCUGCUCAAGUAUGUACCGUGGUCAAACAACUAACUGCAUAUCAGGAAGGAUUUC AUCAACUUUGCUUACAGCGAAAGGAUGACUAAACAAUACUGCAUAAGAAAGCAGGAAAAUUUGACAAAAG AGUUUAAGAGACUAUUAUCAGUAUUUUGGGCAGCACAGUCAAAUGUUAGGCUAUGUAAAUAAUAGCAAAU AGAACUUCUGUCAAUAACUUUUUUCUCAGUUAUCUUUGUUUUUUUUGAGGAGUGUAAUAUGUACUUAGAA UAAUUUUUCAAGUUCUGUGGUUAUUCUAUGAACAAAAAGGGAUAUGAAAGAAGAAAGGCUAUGAAACACA AUACUCUAAAGGUAAGUCUUAUCCUUGUUGGCAUGUGAGUCAAGAUGAAUGAAAAACAUUUGUAGAAAUG AAUAGCCUACAUAUAUUUUUAAUCCCAUGGCAUUUUAUAGGCACAUAUAAAUUGGUUCUGAUGCAAUGUA GAAUAAAUUUAAGCACCUCAAAAUAUAGAAUAACCUCUGUACAUUGAUAAAAAGUCAAGCUCCCUAAUAA UGCAUUUUAACUAAACAUUCAUAACUAAUGUCCAUUACUUCUAUCAAGCAUUUCAUUGCAAAGCCAAGGC AAAUACUUUUGUUGAUUUCCAAAUAAUAGAUAAAUAAUAGAUUUUUCUGUAUGCGUGUGUUGUUUAUUUA CAUGGGAAACAAGACUAAUUACAAUCCUGUGAAAAGAUGACAAGGCAGAUGUAAAAGUUUUCUACAUGGU CUUCUGAUUUAUUAAAAACCAAAAAACUGAAUCACUUUUGUAUGCUAUAAUCAAUAAUUCCUACAGAGUU CUCUUACGAUUCUUAAAGAAUCAUCAGUGCAAAAAUAACCAUCUGCUAAUGCUGCCCACCUUUCUUAGGA AAUCAGAGUUAGUGACUGCACUGCCUUCGAGAAUAUUUUGAUAAAAAGGAUUUUGGCAUAGACUUCCUCA AAAGAGUUUUAUUAACACAAAUAAAUCACUUUCACAGGCUGUAAACAAUAUAUCAAAAAUGAAGCUCUAU UUUUUGCUUUCCUUGCUGUCUUUCCCUUUGUCUUCCUUUUCUGUUCUGUCUUGUUCAUAUUUCUCUUUGU CUGGCUUUGUUACACUAUCAUAUGAAGGUGGAGAGGUGGUGGAUGAAGUGGCAUCUGUUUUUUCUGGACU UGAGUUCUCAUUAACAUUAUCAAAAGCCAUAUCUUUUUUAUUGAGUAAAUCAUCAUCUCUGUCUCCAUCU UUUAUGUAUAUACUUGAUAUAUUUUUGACAUUUUGCCUUAAGCGGUAACGUCUAUAAGCACGCUGAAUGA CAGUAGCAGACACAUCCUCUUGUUUCCGUUUUAGUGUGGUUGUGAUGGGUUCAUAGGACACUUUGGAAGG AUUUGCAGACAUGAACCUUUCUUCCAUCUGUGAACGAAGAGAAUCCAUCUCCCCACUCUCACCCAAAACA CGCUUUGUAAAAGCAAAUAAGAUGUCAAGACAAUGGAUCCGGUCACCACUAACCAUGGGCAGAUCCAUGG CAAUGAGCUGGACUUUGUUGGGUUUUGCUAUGAGAAGAGGAGGAUCCAGGGCAGCUGCAAAAUCAGAGAG UUUAGAGAACUCUAUAAACUGGGUCGCAUCGGGAUCAAACUUCUCCCAAACCUCAUAGAACAUCUCAAAG UCAUCCUCACUCAGAGGUUCAGUACUUUCUUCAGUGGCAACACUAAAAUUCUCCAGUAUGACUGCAAUGU ACAUGUUCACCACAACCAGGAAGGAUAUGAUGAUAUAACUAACAAAGUAGAAUAUUCCAACAGAUGGGUU ACCACAGUCUCCUUCAACUGAACUUCCAGGAUGAACUUUUUUUGGGUCACAGUCGGGUGGCUUACUGUUA AGAAUAGGUGCUAGCAAUCCAUCCCAGCCAGCAGAGGUUGUAAUUUGGAACAGGCAAAUCAUACUGUUGC CAAAGGUCUCAAAAUUGAACAUGUCAUUAAUUCCAUCUUCCUUUUUAACAUAGGCAAAGUUGGACAUUCC AAAGAUGGCGUAGAUGAACAUGACCAGGAAGAGCAGGAGGCCGAUGUUAAACAACGCAGGAAGGGACAUC AUCAAAGCAAAGAGCAGCGUGCGGAUCCCCUUUGCUCCUUUGACUAGACGUAGGAUUCGGCCAAUCCUGG CAAGACGGAUCACUCGGAACAGGGUAGGGGACACAAAAUACGUUUCAAUCAAAUCAGCUAGAAACAUACC UACAAUGGAGAUAAUCACAACCACAAAAUCAAAAAUAUUCCAUCCUACAGUGAAGUAGUAGUGUCUGAGG GAGAUCAGUUUUAGCACACAUUCUCCAGUGAAAAGGAUUAUAAAAACCACAUUUAUCCAAUAUAAAACUU CAGUCAUAUGUUGACUUUGACCCUCCUUUUCUACCAUCAUGGUUACCAUGUUGAGACAGAUAAGAACCAU GAUACUAAUAUCAAAGGCUUGAUUUGUCACUAGGUCAAAUAUACAUCCUUGGAUUUUGUUCCCUGGUCGA GGAAUUGGCUUUUGUGGCUUCUUGGACCCCAGCUUUUUCAUUGCAUUAUAGUAUUUCUUCUGUUCUUCUG UCAUAAAGAUGUCUUGACCUCCAAGCUUCUUUUUCUGUUGGUUGAAAUUAUCUAUGAUGACACCAAUGAA CAAGUUCAAAGUGAAGAAUGACCCAAAGAUGAUAAAGACGACAAAAUAAAUAUACAUGUAGAGGCUAUAU UCAUAUUUGGGCUGCUUGUCUACAUUAACAGAAUCCACUGCUGCAUACAUAAUAAUCGUCCAUCCCUUAA AAGUUGCAACUUGAAGCAGAGAUAGGUAACCAAGUCCGACAUUAUCAAAGUUCACUUUCAGGUUUUUCCA UCGCACAUUUUGACUAACAUUCAUAAGGGCAAAACAUUCGGAACGAUUUGGAACUUGACUUGCAGGAAAC CGUGACCCAUCUGUGGUGUUAAUACACUCAUAGAACUUGCCAGCAAACAAAUUUACUCCCAUGAUGCUGA AUAUCAGCCAGAAUAUAAGACACACAAGUAGCACAUUCAUGAUGGAAGGAAUUGCUCCUAUGAGUGCAUU CACAACGACCCUCAUUCCUUCAAAUCUAGAUAAGGCUCUUAGAGGUCUUAAAGCUCUCAGUGUCCGAAGG GAUUUAAUGGGGCCAAGAUCUGAGUAGCCAAGAGUGUUUGCCACUAAAGUAACCAAAGAAACAUCAACAA UUAGGAAAUCCAGCCAACACCAGGCAUUGGUGAAAUAUGUUUUAUAACCAUAUGCUAUCCAUUUUAGAAG CAUUUCCAGAAUGAAGAUGUAAGUGAAGAUCUUGUCUGCAUACUCCAGGAUAAUCUUAAUGGUCUUUUUC CUUUCAAUAUAAAUAUCUUCAAAAGCCAGGGCACCACUGCUGAGCAGGAUCAUGAGGACAAUGAAGCUUU CAAACCAACUGUGUUCAACAAUCUUGUAGCAGGUUUUCCUGAUGUUCCACCAGAUUUUUCCUUUCCCUGA CUCUAUGUUAACUUGGCAGCAUGAGAACCUCCGUACACAACCAUCUGUGAAACAGGCCUCUGGCUCAUCG GAAUUCAUAGGUUCAGCCUCUGCUUCUUCUCCUUCUCCAGGCAAAGGGUUAUCAACUGUGCUGCACUCUG AGGAGCUUGACCGGUUUAAUCUCACUUUGCUGUAUUCACUAUCCGAAUCACUGCUAAGUUCCUCAGCAUU CAUAUUUUCCAAAUCGGAUUCCCCAGGUGCAAUUGGCACUGUCACUGUGAGGCUGGGAUUGUGAAUAAAU GAUUGACCAUCACUGUCUUCCAUCAAGUGUUUGUCCACGCUGCUUCCAAAACCACUGAUUUUAUCUUUUU CCUUGAGGAAAUUGUGACCUUUGCUCAUUUCAGCAAGUGUAUGGUUAGAAAUAUAGUUUUCCUUCUUAGU AUUCAGAUCUUCUGCUUGUCUUAUCUCCCUGGAAAUCUUUGGCUUUUUGGAAAAUGCUUUUAGAAUAAAU UCACGUAAGGUUUGUUUCACAUAAUUUAUUCCCUUUUUAAUUCUAGUCACUGCAAUCUGGAGGUUGUUUG CAUCAGGGUCUUCUUCAAUUGCUGUAAGAUUGUCUGAACUAAAUGAGCUCAAUAAUAAGGCCAGAAAUAG GUUUAGGACCACCAGGUUUCCAAUGACCAUGACCAUCAUGUAAACAAUAAGGCACAUAGCUUGACCAGCG ACCUCCAUACAGUCCCACAUGGUCUCUAUCCACUCUCCACACAGCACGCGGAACACAAUCAGGAAGGAGU GGAAGAAGUCGUUCAUGUGCCACCGUGGGAGCGUACAGUCAUCAUUGAUCUUGCAGACACAUUCUUUGUA GCUCUUACCAAAGAGCUGCAUGCCGACCACAGCAAAAAUGAAGACGAUGAUGGCCAACACUAAGGUGAGG UUACCUAGAGCCCCUACUGAGUUACCAAUGAUCUUAAUCAGCAUGUUCAAUGUUGGCCAGGAUUUUGCCA ACUUGAAGACUCGGAGCAGUCUGAAUGAUCGCAGAACUGACAAUCCUUCCACAUCUGCUAGAAAGAGCUC CACUAAACUUAAAGUCACAAUAAGGCUGUCAAAAAUAUUCCAGCCUACUUGGAAAUACUCAUAUGGAUCC AUGGCAAUCAGUUUUAAUACCAUUUCAGCUGCAAAGAUUCCAGUAAAGACCAAAUUUCCUAUAGCAAGUA CAUUUUUGAAUUCCUCAGUCAUUGGGUGGUGUUCCAUAGCCAUAAAUAAUGUGUUUAAAACUAUGCAAAU GGUAAUUGCAAGAUCUACAAAAGGAUCCAUUACAAUAAAAUAGAUACACUUUUUGAAUUUUAUCCAAUAU GGAGAGCAAUUCCAGAUCAAGAAUUUGUGUGCAAAUCUGUACCACCAAGGUGGACAUUUUUGUCUGGACU CUUCAAGUUCUUCCACAGUGUUUGUUAAUAUGCUUGCUCUACUCAUUGCUCUCUGUCUGAGGUUGGGAUC AUUCAGCAUAUCCUCUGAAAGGAGAUAGGAACUACAACGCCUUUUCUUGUGUAUUUGAUUGGUCGUGCCC UCUGGCAGAAGCUGUCCAUUGGGGAGCAUGAGGGCUGAGCGUCCAUCAACCAGGGAGACCACACCGUUGC AGUCCACAGCACUGUGCAUUUUCCCGUUCACCGGCAGCAUUGGUGGGGACCUACUGGCUUGGCUGAUGUU ACUGCUGCGUCGCUCCUGGGGUCUGUGGGGCACAAACAGUGAGCCCCUUCUGCUCUCAUUGUCUCCAAAA AUGCUGUGCUCAUCAUCGGCAAAUUCAGUCUCAGAUCCUAUAUCUCUUCCUCUGCCUUUGAAACUAAAAA GACUUGUUCUGCUGCUUCGCCUUGCAGAAAACAAGGAGCCACGAAUGCUGAGUGGUGACUGAUUGGGGGU AGACAACCUCUUUUCAUGUGCUCGCCUAUGCCCUUCGACACCAAGGUGGAAACUUUUUCUUCUGAUGCUG UCCUCUGAUUCUGAUUUCGACAAUUUCUCAGCAUCUCCCUUUUCCUCUCCACUGGAGAGCUUCUUUUGAU UCUUUUUCUUUCUUCUGUUUCUUCUUUCUUUAGCACUUUUAGAGCUCAGUUUGGAUGUUUCAGAAGAACU CUCUGAGAGGCCCAUAAUUCUGCUUCUCCUAAUACUUGUAUAUUCAGCCGCUGCCGCUGCAAUUGCCUCA GCUUCUUCUUGCUCUUUUUUAAGACGGUCUAACAUCUGUUGAAAUUCUAAUUCUUUCUGUUUAGCUUCUU CAAUGUUUGCCUGGUUCUGUUCUUCAUAUGCCAUGGCAACCACAGCCAGGAUCAAGUUUAUUAGAUAAAA GGAGCCCAGGAAAAUCACUACGACAAAGAAGAUCAUGUAGGUUUUGCCAGCAGCACGCAGCGUCUGUUGG UAAAGGUUUUCCCAGUAAUCUUGGGUCAUUAGCCUAAACAAGGCUAAGAAGGCCCAGCUGAAAGUGUCAA AGCUCGUGUAGCCAUAAUCAGGGUUUCUGCCAAUUUUCACACAGGUGUACCCCUCUGGACACUGACCUGA AUCUGUGCUGAAACCACAAAGGAGAGCAUCUUUGGAUCCUUCCAAGUAAUAAAAAUAUUUUCUAAAGUCU UCUUCACUCUCUAGGGUAUUCAUUAUGCUUUCUAAUGUUUCAUUAUUUUCAAGUGAAUUUCGAAAACAUU UAUGCUUCAGGUUUCCCAUGAACAGCUGUAGUCCAAUUAGUGCAAACACACUCAGACAGAACACAGUCAG GAUCAUGACAUCAGAAAGCUUCUUCACUGACUGGAUCAAAGCCCCUACAAUUGUCUUCAGGCCUGGGAUU ACAGAAAUAGUUUUCAAAGCUCUCAAUACUCUGAAAGUUCGAAGAGCUGAAACAUUGCCUAGGUUUACAA AUUCUGUUAAAUACGCAAAAACAAUGACGACAAAAUCCAGCCAGUUCCACGGGUCACGAAGAAAAGUGAA UUCUCCUACACAGAAGCCUCUUGCAAGGAUUUUUACAAGUGAUUCAAAAGUAUAUAUUCCAGUAAAAGUG UACUCGACAUUUUUGGUCCAGUCCGGUGGGUUAUUCAUGGUCAUAAAUAUGCAGUUUGUCAGAAUAGUGC ACAUGAUGAGCAUGCUGAAUAAGGAGUGUACUAAAAUCUUAAUAGAUAUUCUUCUUAGAGGACUGAAAGG AGAAAGCAUAUAUAAAGCAGGUGUGGCAUUGAAACGGAAGAUUGUUUUCCCUUUGUUCAAUACUAUGAAA GUCUUUUUGUCUGCAUAGUAGGGGUCCAAGUCCUCCAGGGGCUCUGACACCAUGCCGGGAGGAAUGUCCC CAUAGAUGAAGGGCAGCUGUUUGCCAGCUUCCAAGUCACUGCUUGGCUUUGGGGCUUCUUCAUCAUCAUC UUUCUUUUCUUCUUUGGGUUCCUUUGAUUUUCUUUCAGCAAUGCGUUGUUCAAUGAGGGCAAGAGACUGU UUUGUGAAAUGGACAAAGCUCUGAGGUCCUGGGGGAGGCAACAUUGCCAUCUUUUCAUCCUGUAUAUUUU AAUUCCUCUUCAGCUCCUCACAUAAGAGGCUUGCAACCUAGCCCGCCGAUCAUCCCCACCCAGUGCACCU GCAGAAUCUGGCUCCAGGAGAGGGCGCGGGCCUCUCCUUCCCCGGCGCUCUCUCAGGGCUGCUUCUUUUU CUCUGGGCUCCUGUUGCUCAGGGGACGCCUGCCGCUAGCAGCCACUGGCACCCAGGCUAGCCCAGCCUCA GCCGAGCUGGCGGAAUUGGAAAGCCGACAGCCGCCGCUGGAGCGCUGGCGACCGCCUGCAAGCAGACUGC GCCCCUCCUGCCAGGGCGCGCCCGUGGAGGUAGCAGCCCCG

A human SCN9A mRNA may have the sequence of SEQ ID NO: 4001 provided herein.

Homo sapiens Sodium Channel, Voltage Gated, Type IX Alpha Subunit (SCN9A), Transcript Variant 2, mRNA

(SEQ ID NO: 4001) AGTCTGCTTGCAGGCGGTCGCCAGCGCTCCAGCGGCGGCTGTCGGCTTTCCAATTCCGCCAGCTCGGCTG AGGCTGGGCTAGCCTGGGTGCCAGTGGCTGCTAGCGGCAGGCGTCCCCTGAGCAACAGGAGCCCAGAGAA AAAGAAGCAGCCCTGAGAGAGCGCCGGGGAAGGAGAGGCCCGCGCCCTCTCCTGGAGCCAGATTCTGCAG GTGCACTGGGTGGGGATGATCGGCGGGCTAGGTTGCAAGCCTCTTATGTGAGGAGCTGAAGAGGAATTAA AATATACAGGATGAAAAGATGGCAATGTTGCCTCCCCCAGGACCTCAGAGCTTTGTCCATTTCACAAAAC AGTCTCTTGCCCTCATTGAACAACGCATTGCTGAAAGAAAATCAAAGGAACCCAAAGAAGAAAAGAAAGA TGATGATGAAGAAGCCCCAAAGCCAAGCAGTGACTTGGAAGCTGGCAAACAGCTGCCCTTCATCTATGGG GACATTCCTCCCGGCATGGTGTCAGAGCCCCTGGAGGACTTGGACCCCTACTATGCAGACAAAAAGACTT TCATAGTATTGAACAAAGGGAAAACAATCTTCCGTTTCAATGCCACACCTGCTTTATATATGCTTTCTCC TTTCAGTCCTCTAAGAAGAATATCTATTAAGATTTTAGTACACTCCTTATTCAGCATGCTCATCATGTGC ACTATTCTGACAAACTGCATATTTATGACCATGAATAACCCACCGGACTGGACCAAAAATGTCGAGTACA CTTTTACTGGAATATATACTTTTGAATCACTTGTAAAAATCCTTGCAAGAGGCTTCTGTGTAGGAGAATT CACTTTTCTTCGTGACCCGTGGAACTGGCTGGATTTTGTCGTCATTGTTTTTGCGTATTTAACAGAATTT GTAAACCTAGGCAATGTTTCAGCTCTTCGAACTTTCAGAGTATTGAGAGCTTTGAAAACTATTTCTGTAA TCCCAGGCCTGAAGACAATTGTAGGGGCTTTGATCCAGTCAGTGAAGAAGCTTTCTGATGTCATGATCCT GACTGTGTTCTGTCTGAGTGTGTTTGCACTAATTGGACTACAGCTGTTCATGGGAAACCTGAAGCATAAA TGTTTTCGAAATTCACTTGAAAATAATGAAACATTAGAAAGCATAATGAATACCCTAGAGAGTGAAGAAG ACTTTAGAAAATATTTTTATTACTTGGAAGGATCCAAAGATGCTCTCCTTTGTGGTTTCAGCACAGATTC AGGTCAGTGTCCAGAGGGGTACACCTGTGTGAAAATTGGCAGAAACCCTGATTATGGCTACACGAGCTTT GACACTTTCAGCTGGGCCTTCTTAGCCTTGTTTAGGCTAATGACCCAAGATTACTGGGAAAACCTTTACC AACAGACGCTGCGTGCTGCTGGCAAAACCTACATGATCTTCTTTGTCGTAGTGATTTTCCTGGGCTCCTT TTATCTAATAAACTTGATCCTGGCTGTGGTTGCCATGGCATATGAAGAACAGAACCAGGCAAACATTGAA GAAGCTAAACAGAAAGAATTAGAATTTCAACAGATGTTAGACCGTCTTAAAAAAGAGCAAGAAGAAGCTG AGGCAATTGCAGCGGCAGCGGCTGAATATACAAGTATTAGGAGAAGCAGAATTATGGGCCTCTCAGAGAG TTCTTCTGAAACATCCAAACTGAGCTCTAAAAGTGCTAAAGAAAGAAGAAACAGAAGAAAGAAAAAGAAT CAAAAGAAGCTCTCCAGTGGAGAGGAAAAGGGAGATGCTGAGAAATTGTCGAAATCAGAATCAGAGGACA GCATCAGAAGAAAAAGTTTCCACCTTGGTGTCGAAGGGCATAGGCGAGCACATGAAAAGAGGTTGTCTAC CCCCAATCAGTCACCACTCAGCATTCGTGGCTCCTTGTTTTCTGCAAGGCGAAGCAGCAGAACAAGTCTT TTTAGTTTCAAAGGCAGAGGAAGAGATATAGGATCTGAGACTGAATTTGCCGATGATGAGCACAGCATTT TTGGAGACAATGAGAGCAGAAGGGGCTCACTGTTTGTGCCCCACAGACCCCAGGAGCGACGCAGCAGTAA CATCAGCCAAGCCAGTAGGTCCCCACCAATGCTGCCGGTGAACGGGAAAATGCACAGTGCTGTGGACTGC AACGGTGTGGTCTCCCTGGTTGATGGACGCTCAGCCCTCATGCTCCCCAATGGACAGCTTCTGCCAGAGG TGATAATAGATAAGGCAACTTCTGATGACAGCGGCACGACCAATCAAATACACAAGAAAAGGCGTTGTAG TTCCTATCTCCTTTCAGAGGATATGCTGAATGATCCCAACCTCAGACAGAGAGCAATGAGTAGAGCAAGC ATATTAACAAACACTGTGGAAGAACTTGAAGAGTCCAGACAAAAATGTCCACCTTGGTGGTACAGATTTG CACACAAATTCTTGATCTGGAATTGCTCTCCATATTGGATAAAATTCAAAAAGTGTATCTATTTTATTGT AATGGATCCTTTTGTAGATCTTGCAATTACCATTTGCATAGTTTTAAACACATTATTTATGGCTATGGAA CACCACCCAATGACTGAGGAATTCAAAAATGTACTTGCTATAGGAAATTTGGTCTTTACTGGAATCTTTG CAGCTGAAATGGTATTAAAACTGATTGCCATGGATCCATATGAGTATTTCCAAGTAGGCTGGAATATTTT TGACAGCCTTATTGTGACTTTAAGTTTAGTGGAGCTCTTTCTAGCAGATGTGGAAGGATTGTCAGTTCTG CGATCATTCAGACTGCTCCGAGTCTTCAAGTTGGCAAAATCCTGGCCAACATTGAACATGCTGATTAAGA TCATTGGTAACTCAGTAGGGGCTCTAGGTAACCTCACCTTAGTGTTGGCCATCATCGTCTTCATTTTTGC TGTGGTCGGCATGCAGCTCTTTGGTAAGAGCTACAAAGAATGTGTCTGCAAGATCAATGATGACTGTACG CTCCCACGGTGGCACATGAACGACTTCTTCCACTCCTTCCTGATTGTGTTCCGCGTGCTGTGTGGAGAGT GGATAGAGACCATGTGGGACTGTATGGAGGTCGCTGGTCAAGCTATGTGCCTTATTGTTTACATGATGGT CATGGTCATTGGAAACCTGGTGGTCCTAAACCTATTTCTGGCCTTATTATTGAGCTCATTTAGTTCAGAC AATCTTACAGCAATTGAAGAAGACCCTGATGCAAACAACCTCCAGATTGCAGTGACTAGAATTAAAAAGG GAATAAATTATGTGAAACAAACCTTACGTGAATTTATTCTAAAAGCATTTTCCAAAAAGCCAAAGATTTC CAGGGAGATAAGACAAGCAGAAGATCTGAATACTAAGAAGGAAAACTATATTTCTAACCATACACTTGCT GAAATGAGCAAAGGTCACAATTTCCTCAAGGAAAAAGATAAAATCAGTGGTTTTGGAAGCAGCGTGGACA AACACTTGATGGAAGACAGTGATGGTCAATCATTTATTCACAATCCCAGCCTCACAGTGACAGTGCCAAT TGCACCTGGGGAATCCGATTTGGAAAATATGAATGCTGAGGAACTTAGCAGTGATTCGGATAGTGAATAC AGCAAAGTGAGATTAAACCGGTCAAGCTCCTCAGAGTGCAGCACAGTTGATAACCCTTTGCCTGGAGAAG GAGAAGAAGCAGAGGCTGAACCTATGAATTCCGATGAGCCAGAGGCCTGTTTCACAGATGGTTGTGTATG GAGGTTCTCATGCTGCCAAGTTAACATAGAGTCAGGGAAAGGAAAAATCTGGTGGAACATCAGGAAAACC TGCTACAAGATTGTTGAACACAGTTGGTTTGAAAGCTTCATTGTCCTCATGATCCTGCTCAGCAGTGGTG CCCTGGCTTTTGAAGATATTTATATTGAAAGGAAAAAGACCATTAAGATTATCCTGGAGTATGCAGACAA GATCTTCACTTACATCTTCATTCTGGAAATGCTTCTAAAATGGATAGCATATGGTTATAAAACATATTTC ACCAATGCCTGGTGTTGGCTGGATTTCCTAATTGTTGATGTTTCTTTGGTTACTTTAGTGGCAAACACTC TTGGCTACTCAGATCTTGGCCCCATTAAATCCCTTCGGACACTGAGAGCTTTAAGACCTCTAAGAGCCTT ATCTAGATTTGAAGGAATGAGGGTCGTTGTGAATGCACTCATAGGAGCAATTCCTTCCATCATGAATGTG CTACTTGTGTGTCTTATATTCTGGCTGATATTCAGCATCATGGGAGTAAATTTGTTTGCTGGCAAGTTCT ATGAGTGTATTAACACCACAGATGGGTCACGGTTTCCTGCAAGTCAAGTTCCAAATCGTTCCGAATGTTT TGCCCTTATGAATGTTAGTCAAAATGTGCGATGGAAAAACCTGAAAGTGAACTTTGATAATGTCGGACTT GGTTACCTATCTCTGCTTCAAGTTGCAACTTTTAAGGGATGGACGATTATTATGTATGCAGCAGTGGATT CTGTTAATGTAGACAAGCAGCCCAAATATGAATATAGCCTCTACATGTATATTTATTTTGTCGTCTTTAT CATCTTTGGGTCATTCTTCACTTTGAACTTGTTCATTGGTGTCATCATAGATAATTTCAACCAACAGAAA AAGAAGCTTGGAGGTCAAGACATCTTTATGACAGAAGAACAGAAGAAATACTATAATGCAATGAAAAAGC TGGGGTCCAAGAAGCCACAAAAGCCAATTCCTCGACCAGGGAACAAAATCCAAGGATGTATATTTGACCT AGTGACAAATCAAGCCTTTGATATTAGTATCATGGTTCTTATCTGTCTCAACATGGTAACCATGATGGTA GAAAAGGAGGGTCAAAGTCAACATATGACTGAAGTTTTATATTGGATAAATGTGGTTTTTATAATCCTTT TCACTGGAGAATGTGTGCTAAAACTGATCTCCCTCAGACACTACTACTTCACTGTAGGATGGAATATTTT TGATTTTGTGGTTGTGATTATCTCCATTGTAGGTATGTTTCTAGCTGATTTGATTGAAACGTATTTTGTG TCCCCTACCCTGTTCCGAGTGATCCGTCTTGCCAGGATTGGCCGAATCCTACGTCTAGTCAAAGGAGCAA AGGGGATCCGCACGCTGCTCTTTGCTTTGATGATGTCCCTTCCTGCGTTGTTTAACATCGGCCTCCTGCT CTTCCTGGTCATGTTCATCTACGCCATCTTTGGAATGTCCAACTTTGCCTATGTTAAAAAGGAAGATGGA ATTAATGACATGTTCAATTTTGAGACCTTTGGCAACAGTATGATTTGCCTGTTCCAAATTACAACCTCTG CTGGCTGGGATGGATTGCTAGCACCTATTCTTAACAGTAAGCCACCCGACTGTGACCCAAAAAAAGTTCA TCCTGGAAGTTCAGTTGAAGGAGACTGTGGTAACCCATCTGTTGGAATATTCTACTTTGTTAGTTATATC ATCATATCCTTCCTGGTTGTGGTGAACATGTACATTGCAGTCATACTGGAGAATTTTAGTGTTGCCACTG AAGAAAGTACTGAACCTCTGAGTGAGGATGACTTTGAGATGTTCTATGAGGTTTGGGAGAAGTTTGATCC CGATGCGACCCAGTTTATAGAGTTCTCTAAACTCTCTGATTTTGCAGCTGCCCTGGATCCTCCTCTTCTC ATAGCAAAACCCAACAAAGTCCAGCTCATTGCCATGGATCTGCCCATGGTTAGTGGTGACCGGATCCATT GTCTTGACATCTTATTTGCTTTTACAAAGCGTGTTTTGGGTGAGAGTGGGGAGATGGATTCTCTTCGTTC ACAGATGGAAGAAAGGTTCATGTCTGCAAATCCTTCCAAAGTGTCCTATGAACCCATCACAACCACACTA AAACGGAAACAAGAGGATGTGTCTGCTACTGTCATTCAGCGTGCTTATAGACGTTACCGCTTAAGGCAAA ATGTCAAAAATATATCAAGTATATACATAAAAGATGGAGACAGAGATGATGATTTACTCAATAAAAAAGA TATGGCTTTTGATAATGTTAATGAGAACTCAAGTCCAGAAAAAACAGATGCCACTTCATCCACCACCTCT CCACCTTCATATGATAGTGTAACAAAGCCAGACAAAGAGAAATATGAACAAGACAGAACAGAAAAGGAAG ACAAAGGGAAAGACAGCAAGGAAAGCAAAAAATAGAGCTTCATTTTTGATATATTGTTTACAGCCTGTGA AAGTGATTTATTTGTGTTAATAAAACTCTTTTGAGGAAGTCTATGCCAAAATCCTTTTTATCAAAATATT CTCGAAGGCAGTGCAGTCACTAACTCTGATTTCCTAAGAAAGGTGGGCAGCATTAGCAGATGGTTATTTT TGCACTGATGATTCTTTAAGAATCGTAAGAGAACTCTGTAGGAATTATTGATTATAGCATACAAAAGTGA TTCAGTTTTTTGGTTTTTAATAAATCAGAAGACCATGTAGAAAACTTTTACATCTGCCTTGTCATCTTTT CACAGGATTGTAATTAGTCTTGTTTCCCATGTAAATAAACAACACACGCATACAGAAAAATCTATTATTT ATCTATTATTTGGAAATCAACAAAAGTATTTGCCTTGGCTTTGCAATGAAATGCTTGATAGAAGTAATGG ACATTAGTTATGAATGTTTAGTTAAAATGCATTATTAGGGAGCTTGACTTTTTATCAATGTACAGAGGTT ATTCTATATTTTGAGGTGCTTAAATTTATTCTACATTGCATCAGAACCAATTTATATGTGCCTATAAAAT GCCATGGGATTAAAAATATATGTAGGCTATTCATTTCTACAAATGTTTTTCATTCATCTTGACTCACATG CCAACAAGGATAAGACTTACCTTTAGAGTATTGTGTTTCATAGCCTTTCTTCTTTCATATCCCTTTTTGT TCATAGAATAACCACAGAACTTGAAAAATTATTCTAAGTACATATTACACTCCTCAAAAAAAACAAAGAT AACTGAGAAAAAAGTTATTGACAGAAGTTCTATTTGCTATTATTTACATAGCCTAACATTTGACTGTGCT GCCCAAAATACTGATAATAGTCTCTTAAACTCTTTTGTCAAATTTTCCTGCTTTCTTATGCAGTATTGTT TAGTCATCCTTTCGCTGTAAGCAAAGTTGATGAAATCCTTCCTGATATGCAGTTAGTTGTTTGACCACGG TACATACTTGAGCAGATAATAACTTGGGCACAGTATTTATTGCATCACTTGTATACAATCCCGTGTTTGG CAAGCTTTCAAATCATGTAATATGACAGACTTTACACAGATATGTGTTTAGTATGAATAAAAAAGCATTG AAATAGGGATTCTTGCCAACTTGCTCTCTTGCCACCAACTTACTTTCCTAAATTATGGAAGTAATCTTTT TTGGATATACTTCAATGTATACAATGAGGAAGATGTCACCTTCTCCTTAAAATTCTATGATGTGAAATAT ATTTTGCCTCAATCAACACAGTACCATGGGCTTCTAATTTATCAAGCACATATTCATTTTGCATTAGCTG TAGACATCTAGTTTTTTGAAAACACCTATTAATAGTAATTTGAAAAGAAATAACCATAATGCTTTTTTTC GTGAGTTTATTTCAGGAATATGAGATCTTTCTTCTATAAAGTTATTCATGCACAGGCAAAAATTGAGCTA CACAGGTAGAATGTAGTTTTACTTAGAAGATTTTTGTGGGAGGTTTTGAAGCAAATATATAAAACAACTT TCACTAATTTGCTTTCCATATTTAAAAAATAATAAATTACATTTATATAATAAATGTTTAAAGCACATAT TTTTTGTTGTTCTGGCAATTTAAAAAGAAAGAGGATTTAAACGTACCTATAGAAACAAAGATTTATGGTT AAAGAATGAGATCAGAAGTCTAGAATGTTTTTAAATTGTGATATATTTTACAACATCCGTTATTACTTTG AGACATTTGTCCTAATCTACGTATAAAACTCAATCTAGGGCTAAAGATTCTTTATACCATCTTAGGTTCA TTCATCTTAGGCTATTTGAACCACTTTTTAATTTAATATGAAAGACACCATGCAGTGTTTTCCGAGACTA CATAGATCATTTTATCACATACCTACCAAGCCTGTTGGAAATAGGTTTTGATAATTTAAGTAGGGACCTA TACAAAATATATTACATTTATCAGATTTTTAAATACATTCAATTAAGAATTTAACATCACCTTAAATTTG AATTCAATCTACCGTTATTTCAAACTCACAAATATAACTGCATTATGAATACTTACATAATGTAGTAAGA CAAGATGTTTGACAGGTTCGTGTGTAATTTTCTATTAATGTTTTTACATTGCCTTGTTTTTATGTAAAAT AAAAAATATGGGCAACTGGTTTGTTAACAACACAATTTCTTCTTAGCATTTCAAAAATATATATAAAGTT GTTCTTTTTCCTATTTCATGAACTATGTTTTTTTTTAAAATAACATGGTTAAGTTTTATATATATTTACG TTTGTTTCAGGAATGTCTACTTGTGACTTTTTATCAATTAAAAATAATATTTGGAAGAAAGAGCTTATTA AGTATAAGCTTGAAGTAAAATTAGACCTCTCTTTCCATGTAGATTACTGTTTGTACTGATGGTTTCACCC TTCAGAAGGCACTGTCATATTAATATTTAAATTTTATAATCGCTGAACTTATTACACCCAACAATACAGA AAGGCAGTTACACTGAAGAACTTAACTTAGAATAAAATGGAAGCAAACAGGTTTTCTAAAAACTTTTTTA AGTGACCAGGTCTCGCTCTGTCACCCAGGCTAGAGTGCAATGGCATGATCATAGCTCTCTGCAGCCTCAA CTCTGGGCTCAAGCAACCCTCCTGCCTCAGCCTCCCAAGTAGCTAAGACTACAGGTACATGCCACCATGC CTGGCTAATATTTAAATTTTTGTAGATAAGGGGTCTTGCTATGTTGCCCAGGCTAGTCTCAAACTCCTGG CTTCAAGTGTTCCTACTGTCATGACCTGCCAACATGCTGGGGTTACAGGCATGAGCCACCATGCCCCAAA CAGGTTTGAACACAAATCTTTCGGATGAAAATTAGAGAACCTAATTTTAGCTTTTTGATAGTTACCTAGT TTGCAAAAGATTTGGGTGACTTGTGAGCTGTTTTTAAATGCTGATTGTTGAACATCACAACCCAAAATAC TTAGCATGATTTTATAGAGTTTTGATAGCTTTATTAAAAAGAGTGAAAATAAAATGCATATGTAAATAAA GCAGTTCTAAATAGCTATTTCAGAGAAATGTTAATAGAAGTGCTGAAAGAAGGGCCAACTAAATTAGGAT GGCCAGGGAATTGGCCTGGGTTTAGGACCTATGTATGAAGGCCACCAATTTTTTAAAAATATCTGTGGTT TATTATGTTATTATCTTCTTGAGGAAAACAATCAAGAATTGCTTCATGAAAATAAATAAATAGCCATGAA TATCATAAAGCTGTTTACATAGGATTCTTTACAAATTTCATAGATCTATGAATGCTCAAAATGTTTGAGT TTGCCATAAATTATATTGTAGTTATATTGTAGTTATACTTGAGACTGACACATTGTAATATAATCTAAGA ATAAAAGTTATACAAAATAAAA

The reverse complement of SEQ ID NO: 4001 is provided as SEQ ID NO: 4002 herein:

(SEQ ID NO: 4002) TTTTATTTTGTATAACTTTTATTCTTAGATTATATTACAATGTGTCAGTCTCAAGTATAACTACAATATA ACTACAATATAATTTATGGCAAACTCAAACATTTTGAGCATTCATAGATCTATGAAATTTGTAAAGAATC CTATGTAAACAGCTTTATGATATTCATGGCTATTTATTTATTTTCATGAAGCAATTCTTGATTGTTTTCC TCAAGAAGATAATAACATAATAAACCACAGATATTTTTAAAAAATTGGTGGCCTTCATACATAGGTCCTA AACCCAGGCCAATTCCCTGGCCATCCTAATTTAGTTGGCCCTTCTTTCAGCACTTCTATTAACATTTCTC TGAAATAGCTATTTAGAACTGCTTTATTTACATATGCATTTTATTTTCACTCTTTTTAATAAAGCTATCA AAACTCTATAAAATCATGCTAAGTATTTTGGGTTGTGATGTTCAACAATCAGCATTTAAAAACAGCTCAC AAGTCACCCAAATCTTTTGCAAACTAGGTAACTATCAAAAAGCTAAAATTAGGTTCTCTAATTTTCATCC GAAAGATTTGTGTTCAAACCTGTTTGGGGCATGGTGGCTCATGCCTGTAACCCCAGCATGTTGGCAGGTC ATGACAGTAGGAACACTTGAAGCCAGGAGTTTGAGACTAGCCTGGGCAACATAGCAAGACCCCTTATCTA CAAAAATTTAAATATTAGCCAGGCATGGTGGCATGTACCTGTAGTCTTAGCTACTTGGGAGGCTGAGGCA GGAGGGTTGCTTGAGCCCAGAGTTGAGGCTGCAGAGAGCTATGATCATGCCATTGCACTCTAGCCTGGGT GACAGAGCGAGACCTGGTCACTTAAAAAAGTTTTTAGAAAACCTGTTTGCTTCCATTTTATTCTAAGTTA AGTTCTTCAGTGTAACTGCCTTTCTGTATTGTTGGGTGTAATAAGTTCAGCGATTATAAAATTTAAATAT TAATATGACAGTGCCTTCTGAAGGGTGAAACCATCAGTACAAACAGTAATCTACATGGAAAGAGAGGTCT AATTTTACTTCAAGCTTATACTTAATAAGCTCTTTCTTCCAAATATTATTTTTAATTGATAAAAAGTCAC AAGTAGACATTCCTGAAACAAACGTAAATATATATAAAACTTAACCATGTTATTTTAAAAAAAAACATAG TTCATGAAATAGGAAAAAGAACAACTTTATATATATTTTTGAAATGCTAAGAAGAAATTGTGTTGTTAAC AAACCAGTTGCCCATATTTTTTATTTTACATAAAAACAAGGCAATGTAAAAACATTAATAGAAAATTACA CACGAACCTGTCAAACATCTTGTCTTACTACATTATGTAAGTATTCATAATGCAGTTATATTTGTGAGTT TGAAATAACGGTAGATTGAATTCAAATTTAAGGTGATGTTAAATTCTTAATTGAATGTATTTAAAAATCT GATAAATGTAATATATTTTGTATAGGTCCCTACTTAAATTATCAAAACCTATTTCCAACAGGCTTGGTAG GTATGTGATAAAATGATCTATGTAGTCTCGGAAAACACTGCATGGTGTCTTTCATATTAAATTAAAAAGT GGTTCAAATAGCCTAAGATGAATGAACCTAAGATGGTATAAAGAATCTTTAGCCCTAGATTGAGTTTTAT ACGTAGATTAGGACAAATGTCTCAAAGTAATAACGGATGTTGTAAAATATATCACAATTTAAAAACATTC TAGACTTCTGATCTCATTCTTTAACCATAAATCTTTGTTTCTATAGGTACGTTTAAATCCTCTTTCTTTT TAAATTGCCAGAACAACAAAAAATATGTGCTTTAAACATTTATTATATAAATGTAATTTATTATTTTTTA AATATGGAAAGCAAATTAGTGAAAGTTGTTTTATATATTTGCTTCAAAACCTCCCACAAAAATCTTCTAA GTAAAACTACATTCTACCTGTGTAGCTCAATTTTTGCCTGTGCATGAATAACTTTATAGAAGAAAGATCT CATATTCCTGAAATAAACTCACGAAAAAAAGCATTATGGTTATTTCTTTTCAAATTACTATTAATAGGTG TTTTCAAAAAACTAGATGTCTACAGCTAATGCAAAATGAATATGTGCTTGATAAATTAGAAGCCCATGGT ACTGTGTTGATTGAGGCAAAATATATTTCACATCATAGAATTTTAAGGAGAAGGTGACATCTTCCTCATT GTATACATTGAAGTATATCCAAAAAAGATTACTTCCATAATTTAGGAAAGTAAGTTGGTGGCAAGAGAGC AAGTTGGCAAGAATCCCTATTTCAATGCTTTTTTATTCATACTAAACACATATCTGTGTAAAGTCTGTCA TATTACATGATTTGAAAGCTTGCCAAACACGGGATTGTATACAAGTGATGCAATAAATACTGTGCCCAAG TTATTATCTGCTCAAGTATGTACCGTGGTCAAACAACTAACTGCATATCAGGAAGGATTTCATCAACTTT GCTTACAGCGAAAGGATGACTAAACAATACTGCATAAGAAAGCAGGAAAATTTGACAAAAGAGTTTAAGA GACTATTATCAGTATTTTGGGCAGCACAGTCAAATGTTAGGCTATGTAAATAATAGCAAATAGAACTTCT GTCAATAACTTTTTTCTCAGTTATCTTTGTTTTTTTTGAGGAGTGTAATATGTACTTAGAATAATTTTTC AAGTTCTGTGGTTATTCTATGAACAAAAAGGGATATGAAAGAAGAAAGGCTATGAAACACAATACTCTAA AGGTAAGTCTTATCCTTGTTGGCATGTGAGTCAAGATGAATGAAAAACATTTGTAGAAATGAATAGCCTA CATATATTTTTAATCCCATGGCATTTTATAGGCACATATAAATTGGTTCTGATGCAATGTAGAATAAATT TAAGCACCTCAAAATATAGAATAACCTCTGTACATTGATAAAAAGTCAAGCTCCCTAATAATGCATTTTA ACTAAACATTCATAACTAATGTCCATTACTTCTATCAAGCATTTCATTGCAAAGCCAAGGCAAATACTTT TGTTGATTTCCAAATAATAGATAAATAATAGATTTTTCTGTATGCGTGTGTTGTTTATTTACATGGGAAA CAAGACTAATTACAATCCTGTGAAAAGATGACAAGGCAGATGTAAAAGTTTTCTACATGGTCTTCTGATT TATTAAAAACCAAAAAACTGAATCACTTTTGTATGCTATAATCAATAATTCCTACAGAGTTCTCTTACGA TTCTTAAAGAATCATCAGTGCAAAAATAACCATCTGCTAATGCTGCCCACCTTTCTTAGGAAATCAGAGT TAGTGACTGCACTGCCTTCGAGAATATTTTGATAAAAAGGATTTTGGCATAGACTTCCTCAAAAGAGTTT TATTAACACAAATAAATCACTTTCACAGGCTGTAAACAATATATCAAAAATGAAGCTCTATTTTTTGCTT TCCTTGCTGTCTTTCCCTTTGTCTTCCTTTTCTGTTCTGTCTTGTTCATATTTCTCTTTGTCTGGCTTTG TTACACTATCATATGAAGGTGGAGAGGTGGTGGATGAAGTGGCATCTGTTTTTTCTGGACTTGAGTTCTC ATTAACATTATCAAAAGCCATATCTTTTTTATTGAGTAAATCATCATCTCTGTCTCCATCTTTTATGTAT ATACTTGATATATTTTTGACATTTTGCCTTAAGCGGTAACGTCTATAAGCACGCTGAATGACAGTAGCAG ACACATCCTCTTGTTTCCGTTTTAGTGTGGTTGTGATGGGTTCATAGGACACTTTGGAAGGATTTGCAGA CATGAACCTTTCTTCCATCTGTGAACGAAGAGAATCCATCTCCCCACTCTCACCCAAAACACGCTTTGTA AAAGCAAATAAGATGTCAAGACAATGGATCCGGTCACCACTAACCATGGGCAGATCCATGGCAATGAGCT GGACTTTGTTGGGTTTTGCTATGAGAAGAGGAGGATCCAGGGCAGCTGCAAAATCAGAGAGTTTAGAGAA CTCTATAAACTGGGTCGCATCGGGATCAAACTTCTCCCAAACCTCATAGAACATCTCAAAGTCATCCTCA CTCAGAGGTTCAGTACTTTCTTCAGTGGCAACACTAAAATTCTCCAGTATGACTGCAATGTACATGTTCA CCACAACCAGGAAGGATATGATGATATAACTAACAAAGTAGAATATTCCAACAGATGGGTTACCACAGTC TCCTTCAACTGAACTTCCAGGATGAACTTTTTTTGGGTCACAGTCGGGTGGCTTACTGTTAAGAATAGGT GCTAGCAATCCATCCCAGCCAGCAGAGGTTGTAATTTGGAACAGGCAAATCATACTGTTGCCAAAGGTCT CAAAATTGAACATGTCATTAATTCCATCTTCCTTTTTAACATAGGCAAAGTTGGACATTCCAAAGATGGC GTAGATGAACATGACCAGGAAGAGCAGGAGGCCGATGTTAAACAACGCAGGAAGGGACATCATCAAAGCA AAGAGCAGCGTGCGGATCCCCTTTGCTCCTTTGACTAGACGTAGGATTCGGCCAATCCTGGCAAGACGGA TCACTCGGAACAGGGTAGGGGACACAAAATACGTTTCAATCAAATCAGCTAGAAACATACCTACAATGGA GATAATCACAACCACAAAATCAAAAATATTCCATCCTACAGTGAAGTAGTAGTGTCTGAGGGAGATCAGT TTTAGCACACATTCTCCAGTGAAAAGGATTATAAAAACCACATTTATCCAATATAAAACTTCAGTCATAT GTTGACTTTGACCCTCCTTTTCTACCATCATGGTTACCATGTTGAGACAGATAAGAACCATGATACTAAT ATCAAAGGCTTGATTTGTCACTAGGTCAAATATACATCCTTGGATTTTGTTCCCTGGTCGAGGAATTGGC TTTTGTGGCTTCTTGGACCCCAGCTTTTTCATTGCATTATAGTATTTCTTCTGTTCTTCTGTCATAAAGA TGTCTTGACCTCCAAGCTTCTTTTTCTGTTGGTTGAAATTATCTATGATGACACCAATGAACAAGTTCAA AGTGAAGAATGACCCAAAGATGATAAAGACGACAAAATAAATATACATGTAGAGGCTATATTCATATTTG GGCTGCTTGTCTACATTAACAGAATCCACTGCTGCATACATAATAATCGTCCATCCCTTAAAAGTTGCAA CTTGAAGCAGAGATAGGTAACCAAGTCCGACATTATCAAAGTTCACTTTCAGGTTTTTCCATCGCACATT TTGACTAACATTCATAAGGGCAAAACATTCGGAACGATTTGGAACTTGACTTGCAGGAAACCGTGACCCA TCTGTGGTGTTAATACACTCATAGAACTTGCCAGCAAACAAATTTACTCCCATGATGCTGAATATCAGCC AGAATATAAGACACACAAGTAGCACATTCATGATGGAAGGAATTGCTCCTATGAGTGCATTCACAACGAC CCTCATTCCTTCAAATCTAGATAAGGCTCTTAGAGGTCTTAAAGCTCTCAGTGTCCGAAGGGATTTAATG GGGCCAAGATCTGAGTAGCCAAGAGTGTTTGCCACTAAAGTAACCAAAGAAACATCAACAATTAGGAAAT CCAGCCAACACCAGGCATTGGTGAAATATGTTTTATAACCATATGCTATCCATTTTAGAAGCATTTCCAG AATGAAGATGTAAGTGAAGATCTTGTCTGCATACTCCAGGATAATCTTAATGGTCTTTTTCCTTTCAATA TAAATATCTTCAAAAGCCAGGGCACCACTGCTGAGCAGGATCATGAGGACAATGAAGCTTTCAAACCAAC TGTGTTCAACAATCTTGTAGCAGGTTTTCCTGATGTTCCACCAGATTTTTCCTTTCCCTGACTCTATGTT AACTTGGCAGCATGAGAACCTCCATACACAACCATCTGTGAAACAGGCCTCTGGCTCATCGGAATTCATA GGTTCAGCCTCTGCTTCTTCTCCTTCTCCAGGCAAAGGGTTATCAACTGTGCTGCACTCTGAGGAGCTTG ACCGGTTTAATCTCACTTTGCTGTATTCACTATCCGAATCACTGCTAAGTTCCTCAGCATTCATATTTTC CAAATCGGATTCCCCAGGTGCAATTGGCACTGTCACTGTGAGGCTGGGATTGTGAATAAATGATTGACCA TCACTGTCTTCCATCAAGTGTTTGTCCACGCTGCTTCCAAAACCACTGATTTTATCTTTTTCCTTGAGGA AATTGTGACCTTTGCTCATTTCAGCAAGTGTATGGTTAGAAATATAGTTTTCCTTCTTAGTATTCAGATC TTCTGCTTGTCTTATCTCCCTGGAAATCTTTGGCTTTTTGGAAAATGCTTTTAGAATAAATTCACGTAAG GTTTGTTTCACATAATTTATTCCCTTTTTAATTCTAGTCACTGCAATCTGGAGGTTGTTTGCATCAGGGT CTTCTTCAATTGCTGTAAGATTGTCTGAACTAAATGAGCTCAATAATAAGGCCAGAAATAGGTTTAGGAC CACCAGGTTTCCAATGACCATGACCATCATGTAAACAATAAGGCACATAGCTTGACCAGCGACCTCCATA CAGTCCCACATGGTCTCTATCCACTCTCCACACAGCACGCGGAACACAATCAGGAAGGAGTGGAAGAAGT CGTTCATGTGCCACCGTGGGAGCGTACAGTCATCATTGATCTTGCAGACACATTCTTTGTAGCTCTTACC AAAGAGCTGCATGCCGACCACAGCAAAAATGAAGACGATGATGGCCAACACTAAGGTGAGGTTACCTAGA GCCCCTACTGAGTTACCAATGATCTTAATCAGCATGTTCAATGTTGGCCAGGATTTTGCCAACTTGAAGA CTCGGAGCAGTCTGAATGATCGCAGAACTGACAATCCTTCCACATCTGCTAGAAAGAGCTCCACTAAACT TAAAGTCACAATAAGGCTGTCAAAAATATTCCAGCCTACTTGGAAATACTCATATGGATCCATGGCAATC AGTTTTAATACCATTTCAGCTGCAAAGATTCCAGTAAAGACCAAATTTCCTATAGCAAGTACATTTTTGA ATTCCTCAGTCATTGGGTGGTGTTCCATAGCCATAAATAATGTGTTTAAAACTATGCAAATGGTAATTGC AAGATCTACAAAAGGATCCATTACAATAAAATAGATACACTTTTTGAATTTTATCCAATATGGAGAGCAA TTCCAGATCAAGAATTTGTGTGCAAATCTGTACCACCAAGGTGGACATTTTTGTCTGGACTCTTCAAGTT CTTCCACAGTGTTTGTTAATATGCTTGCTCTACTCATTGCTCTCTGTCTGAGGTTGGGATCATTCAGCAT ATCCTCTGAAAGGAGATAGGAACTACAACGCCTTTTCTTGTGTATTTGATTGGTCGTGCCGCTGTCATCA GAAGTTGCCTTATCTATTATCACCTCTGGCAGAAGCTGTCCATTGGGGAGCATGAGGGCTGAGCGTCCAT CAACCAGGGAGACCACACCGTTGCAGTCCACAGCACTGTGCATTTTCCCGTTCACCGGCAGCATTGGTGG GGACCTACTGGCTTGGCTGATGTTACTGCTGCGTCGCTCCTGGGGTCTGTGGGGCACAAACAGTGAGCCC CTTCTGCTCTCATTGTCTCCAAAAATGCTGTGCTCATCATCGGCAAATTCAGTCTCAGATCCTATATCTC TTCCTCTGCCTTTGAAACTAAAAAGACTTGTTCTGCTGCTTCGCCTTGCAGAAAACAAGGAGCCACGAAT GCTGAGTGGTGACTGATTGGGGGTAGACAACCTCTTTTCATGTGCTCGCCTATGCCCTTCGACACCAAGG TGGAAACTTTTTCTTCTGATGCTGTCCTCTGATTCTGATTTCGACAATTTCTCAGCATCTCCCTTTTCCT CTCCACTGGAGAGCTTCTTTTGATTCTTTTTCTTTCTTCTGTTTCTTCTTTCTTTAGCACTTTTAGAGCT CAGTTTGGATGTTTCAGAAGAACTCTCTGAGAGGCCCATAATTCTGCTTCTCCTAATACTTGTATATTCA GCCGCTGCCGCTGCAATTGCCTCAGCTTCTTCTTGCTCTTTTTTAAGACGGTCTAACATCTGTTGAAATT CTAATTCTTTCTGTTTAGCTTCTTCAATGTTTGCCTGGTTCTGTTCTTCATATGCCATGGCAACCACAGC CAGGATCAAGTTTATTAGATAAAAGGAGCCCAGGAAAATCACTACGACAAAGAAGATCATGTAGGTTTTG CCAGCAGCACGCAGCGTCTGTTGGTAAAGGTTTTCCCAGTAATCTTGGGTCATTAGCCTAAACAAGGCTA AGAAGGCCCAGCTGAAAGTGTCAAAGCTCGTGTAGCCATAATCAGGGTTTCTGCCAATTTTCACACAGGT GTACCCCTCTGGACACTGACCTGAATCTGTGCTGAAACCACAAAGGAGAGCATCTTTGGATCCTTCCAAG TAATAAAAATATTTTCTAAAGTCTTCTTCACTCTCTAGGGTATTCATTATGCTTTCTAATGTTTCATTAT TTTCAAGTGAATTTCGAAAACATTTATGCTTCAGGTTTCCCATGAACAGCTGTAGTCCAATTAGTGCAAA CACACTCAGACAGAACACAGTCAGGATCATGACATCAGAAAGCTTCTTCACTGACTGGATCAAAGCCCCT ACAATTGTCTTCAGGCCTGGGATTACAGAAATAGTTTTCAAAGCTCTCAATACTCTGAAAGTTCGAAGAG CTGAAACATTGCCTAGGTTTACAAATTCTGTTAAATACGCAAAAACAATGACGACAAAATCCAGCCAGTT CCACGGGTCACGAAGAAAAGTGAATTCTCCTACACAGAAGCCTCTTGCAAGGATTTTTACAAGTGATTCA AAAGTATATATTCCAGTAAAAGTGTACTCGACATTTTTGGTCCAGTCCGGTGGGTTATTCATGGTCATAA ATATGCAGTTTGTCAGAATAGTGCACATGATGAGCATGCTGAATAAGGAGTGTACTAAAATCTTAATAGA TATTCTTCTTAGAGGACTGAAAGGAGAAAGCATATATAAAGCAGGTGTGGCATTGAAACGGAAGATTGTT TTCCCTTTGTTCAATACTATGAAAGTCTTTTTGTCTGCATAGTAGGGGTCCAAGTCCTCCAGGGGCTCTG ACACCATGCCGGGAGGAATGTCCCCATAGATGAAGGGCAGCTGTTTGCCAGCTTCCAAGTCACTGCTTGG CTTTGGGGCTTCTTCATCATCATCTTTCTTTTCTTCTTTGGGTTCCTTTGATTTTCTTTCAGCAATGCGT TGTTCAATGAGGGCAAGAGACTGTTTTGTGAAATGGACAAAGCTCTGAGGTCCTGGGGGAGGCAACATTG CCATCTTTTCATCCTGTATATTTTAATTCCTCTTCAGCTCCTCACATAAGAGGCTTGCAACCTAGCCCGC CGATCATCCCCACCCAGTGCACCTGCAGAATCTGGCTCCAGGAGAGGGCGCGGGCCTCTCCTTCCCCGGC GCTCTCTCAGGGCTGCTTCTTTTTCTCTGGGCTCCTGTTGCTCAGGGGACGCCTGCCGCTAGCAGCCACT GGCACCCAGGCTAGCCCAGCCTCAGCCGAGCTGGCGGAATTGGAAAGCCGACAGCCGCCGCTGGAGCGCT GGCGACCGCCTGCAAGCAGACT

In some embodiments, an iRNA described herein includes at least 15 contiguous nucleotides from 50 one of the sequences provided in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20, and may optionally be coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in SCN9A.

While a target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays described herein or known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., in Tables 2A, 4A, 5A, 6A, 13A, 14A, 15A, 16, 18, and 20, further optimization can be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.

In some embodiments, the disclosure provides an iRNA, e.g., in Tables 2B, 4B, 5B, 6B, 13B, 14B, an 15B, that is un-modified or un-conjugated. In some embodiments, an RNAi agent of the disclosure has a nucleotide sequence as provided in any of Tables 2A, 4A, 5A, 6A, 13A, 14A, 15A, 16, 18, or 20, but lacks one or more ligand or moiety shown in the table. A ligand or moiety (e.g., a lipophilic ligand or moiety) can be included in any of the positions provided in the instant application.

An iRNA as described herein can contain one or more mismatches to the target sequence. In some embodiments, an iRNA as described herein contains no more than 3 mismatches. In some embodiments, when the antisense strand of the iRNA contains mismatches to a target sequence, the area of mismatch is not located in the center of the region of complementarity. In some embodiments, when the antisense strand of the iRNA contains mismatches to the target sequence, the mismatch is restricted to be within the last 5 nucleotides from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide iRNA agent RNA strand which is complementary to a region of SCN9A, the RNA strand generally does not contain any mismatch within the central 13 nucleotides. The methods described herein, or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of SCN9A. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of SCN9A is important, especially if the particular region of complementarity in a SCN9A gene is known to have polymorphic sequence variation within the population.

In some embodiments, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In some embodiments, dsRNAs having at least one nucleotide overhang have superior inhibitory properties relative to their blunt-ended counterparts. In some embodiments, the RNA of an iRNA (e.g., a dsRNA) is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the disclosure may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position, or having an acyclic sugar) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in this disclosure include, but are not limited to, RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, each of which is herein incorporated by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.

In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506. The native phosphodiester backbone can be represented as O—P(O)(OH)—OCH₂—.

Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂.

In other embodiments, an iRNA agent comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides (or nucleosides). In certain embodiments, the sense strand or the antisense strand, or both sense strand and antisense strand, include less than five acyclic nucleotides per strand (e.g., four, three, two or one acyclic nucleotides per strand). The one or more acyclic nucleotides can be found, for example, in the double-stranded region, of the sense or antisense strand, or both strands; at the 5′-end, the 3′-end, both of the 5′ and 3′-ends of the sense or antisense strand, or both strands, of the iRNA agent. In some embodiments, one or more acyclic nucleotides are present at positions 1 to 8 of the sense or antisense strand, or both. In some embodiments, one or more acyclic nucleotides are found in the antisense strand at positions 4 to 10 (e.g., positions 6-8) from the 5′-end of the antisense strand. In some embodiments, the one or more acyclic nucleotides are found at one or both 3′-terminal overhangs of the iRNA agent.

The term “acyclic nucleotide” or “acyclic nucleoside” as used herein refers to any nucleotide or nucleoside having an acyclic sugar, e.g., an acyclic ribose. An exemplary acyclic nucleotide or nucleoside can include a nucleobase, e.g., a naturally occurring or a modified nucleobase (e.g., a nucleobase as described herein). In certain embodiments, a bond between any of the ribose carbons (C1, C2, C3, C4, or C5), is independently or in combination absent from the nucleotide. In some embodiments, the bond between C2-C3 carbons of the ribose ring is absent, e.g., an acyclic 2′-3′-seco-nucleotide monomer. In other embodiments, the bond between C1-C2, C3-C4, or C4-C5 is absent (e.g., a l′-2′, 3′-4′ or 4′-5′-seco nucleotide monomer). Exemplary acyclic nucleotides are disclosed in U.S. Pat. No. 8,314,227, incorporated herein by reference in its entirely. For example, an acyclic nucleotide can include any of monomers D-J in FIGS. 1-2 of U.S. Pat. No. 8,314,227. In some embodiments, the acyclic nucleotide includes the following monomer:

wherein Base is a nucleobase, e.g., a naturally occurring or a modified nucleobase (e.g., a nucleobase as described herein).

In certain embodiments, the acyclic nucleotide can be modified or derivatized, e.g., by coupling the acyclic nucleotide to another moiety, e.g., a ligand (e.g., a GalNAc, a cholesterol ligand), an alkyl, a polyamine, a sugar, a polypeptide, among others.

In other embodiments, the iRNA agent includes one or more acyclic nucleotides and one or more LNAs (e.g., an LNA as described herein). For example, one or more acyclic nucleotides and/or one or more LNAs can be present in the sense strand, the antisense strand, or both. The number of acyclic nucleotides in one strand can be the same or different from the number of LNAs in the opposing strand. In certain embodiments, the sense strand and/or the antisense strand comprises less than five LNAs (e.g., four, three, two or one LNAs) located in the double stranded region or a 3′-overhang. In other embodiments, one or two LNAs are located in the double stranded region or the 3′-overhang of the sense strand. Alternatively, or in combination, the sense strand and/or antisense strand comprises less than five acyclic nucleotides (e.g., four, three, two or one acyclic nucleotides) in the double-stranded region or a 3′-overhang. In some embodiments, the sense strand of the iRNA agent comprises one or two LNAs in the 3′-overhang of the sense strand, and one or two acyclic nucleotides in the double-stranded region of the antisense strand (e.g., at positions 4 to 10 (e.g., positions 6-8) from the 5′-end of the antisense strand) of the iRNA agent.

In other embodiments, inclusion of one or more acyclic nucleotides (alone or in addition to one or more LNAs) in the iRNA agent results in one or more (or all) of: (i) a reduction in an off-target effect; (ii) a reduction in passenger strand participation in RNAi; (iii) an increase in specificity of the guide strand for its target mRNA; (iv) a reduction in a microRNA off-target effect; (v) an increase in stability; or (vi) an increase in resistance to degradation, of the iRNA molecule.

Other modifications include 2′-methoxy (2′-OCH3), 2′-5 aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F) Similar modifications may also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

An iRNA may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine.

Further modified nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these modified nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.

The RNA of an iRNA can also be modified to include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the disclosure may include one or more locked nucleic acids (LNAs) (also referred to herein as “locked nucleotides”). In some embodiments, a locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting, e.g., the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, increase thermal stability, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

Examples of bicyclic nucleosides for use in the polynucleotides of the disclosure include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the disclosure include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)₂—O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(H2)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The contents of each of the foregoing are incorporated herein by reference for the methods provided therein. Representative U.S. patents that teach the preparation of locked nucleic acids include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; 7,399,845, and 8,314,227, each of which is herein incorporated by reference in its entirety. Exemplary LNAs include but are not limited to, a 2′, 4′-C methylene bicyclo nucleotide (see for example Wengel et al., International PCT 5 Publication No. WO 00/66604 and WO 99/14226).

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

A RNAi agent of the disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)-0-2′ bridge. In some embodiments, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”

A RNAi agent of the disclosure may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US 2013/0190383; and WO 2013/036868, the contents of each of which are hereby incorporated herein by reference for the methods provided therein.

In some embodiments, a RNAi agent of the disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039).

Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the contents of each of which are hereby incorporated herein by reference for the methods provided therein.

In other embodiments, the iRNA agents include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in the iRNA molecules can result in enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

Other modifications of a RNAi agent of the disclosure include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of a RNAi agent. Suitable phosphate mimics are disclosed in, for example US 2012/0157511, the contents of which are incorporated herein by reference for the methods provided therein.

iRNA Motifs

In certain aspects of the disclosure, the double-stranded RNAi agents of the disclosure include agents with chemical modifications as disclosed, for example, in WO 2013/075035, the contents of which are incorporated herein by reference for the methods provided therein. As shown herein and in WO 2013/075035, a superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand or antisense strand of an RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand. The RNAi agent may be optionally conjugated with a lipophilic moiety or ligand, e.g., a C16 moiety or ligand, for instance on the sense strand. The RNAi agent may be optionally modified with a (S)-glycol nucleic acid (GNA) modification, for instance on one or more residues of the antisense strand. The resulting RNAi agents present superior gene silencing activity.

In some embodiments, the sense strand sequence may be represented by formula (I):

5′ np-Na-(X X X)i-Nb-Y Y Y-Nb-(Z Z Z)j-Na-nq 3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each Na independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each Nb independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each np and nq independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. In some embodiments, YYY is all 2′-F modified nucleotides.

In some embodiments, the Na and/or Nb comprise modifications of alternating pattern.

In some embodiments, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11,12 or 11, 12, 13) of the sense strand, the count starting from the 1^(st) nucleotide, from the 5′-end; or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end.

In some embodiments, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

5′ np-Na-YYY-Nb-ZZZ-Na-nq 3′  (Ib);

5′ np-Na-XXX-Nb-YYY-Na-nq 3′  (Ic); or

5′ np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq 3′  (Id).

When the sense strand is represented by formula (Ib), Nb represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), Nb represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each Nb independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. In some embodiments, Nb is 0, 1, 2, 3, 4, 5 or 6. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:

5′ n_(p)-N_(a)-YYY-N_(a)-n_(q) 3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

In some embodiments, the antisense strand sequence of the RNAi may be represented by formula (II):

5′ n_(q′)-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(l)-N_(a)′-n_(p)′ 3′  (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each Na′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each Nb′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification;

and

X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one of three identical modification on three consecutive nucleotides.

In some embodiments, the N_(a)′ and/or N_(b)′ comprise modification of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the 1^(st) nucleotide, from the 5′-end; or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end. In some embodiments, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In some embodiments, Y′Y′Y′ motif is all 2′-Ome modified nucleotides.

In on embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both 5 k and l are 1.

The antisense strand can therefore be represented by the following formulas:

5′ n_(q)′-N_(a)′-Z′Z′Z′-Nb′-Y′Y′Y′-N_(a)′-n_(p)′ 3′  (IIb);

5′ n_(q)′-N_(a)′-Y′Y′Y′-Nb′-X′X′X′-n_(p)′ 3′  (IIc); or

5′ n_(q)′-Na′-Z′Z′Z′-Ne′-Y′Y′Y′-Nb′-X′X′X′-N_(a)′-n_(p)′ 3′  (IId).

When the antisense strand is represented by formula (IIb), Nb′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. In some embodiments, Nb is 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may be represented by the formula:

5′ np′-Na′-Y′Y′Y′-Na′-nq′ 3′  (Ia).

When the antisense strand is represented as formula (IIa), each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, HNA, CeNA, GNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In some embodiments, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In some embodiments the antisense strand may Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with an antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, certain RNAi agents for use in the methods of the disclosure may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (III):

sense: 5′ n_(p)-N_(a)-(XXX)i-N_(b)-YYY-N_(b)-(ZZZ)j-N_(a)-n_(q) 3′

antisense: 3′ n_(p)′-Na′-(X′X′X′)k-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′   (III)

wherein,

j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not be present independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modification on three consecutive nucleotides.

In some embodiments, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In some embodiments, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forming a RNAi duplex include the formulas below:

5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′

3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′n_(q)′ 5′   (IIIa)

5′ n_(p)-N_(a)′-Y Y Y-N_(b)-ZZZ-N_(a)-n_(q) 3′

3′ n_(p)-N_(a)′-Y′Y′Y′-Nb′-Z′Z′Z′-Na′-nq′ 5′   (IIIb)

5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′

3′ n_(p)-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-Na′-n_(q)′ 5′   (IIIc)

5′ n_(p)-N_(a)-XXX-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′

3′ n_(p)-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-Na′-n_(q)′ 5′   (IIId)

When the RNAi agent is represented by formula (IIIa), each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each Nb independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each Nb, Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each Nb, Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a), N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b) and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.

In some embodiments, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.

In some embodiments, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications. In some embodiments, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications and np′ >0 and at least one np′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In some embodiments, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties, or one or more GalNAc moieties) attached through a bivalent or trivalent branched linker. In some embodiments, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties, or one or more GalNAc moieties) attached through a bivalent or trivalent branched linker.

In some embodiments, when the RNAi agent is represented by formula (IIIa), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties) attached through a bivalent or trivalent branched linker.

In some embodiments, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In some embodiments, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In some embodiments, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends and are optionally conjugated to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used in the methods of the disclosure. Such publications include WO2007/091269, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520; and U.S. Pat. No. 7,858,769, the contents of each of which are hereby incorporated herein by reference for the methods provided therein. In certain embodiments, the RNAi agents of the disclosure may include GalNAc ligands.

As described in more detail below, the RNAi agent that contains conjugations of one or more carbohydrate moieties to a RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” or two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group. In some embodiments, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin. In some embodiments, the acyclic group is selected from serinol backbone or diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methods of the disclosure is an agent selected from the group of agents listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20. These agents may further comprise a ligand. The ligand can be attached to the sense strand, antisense strand or both strands, at the 3′-end, 5′-end, or both ends. For instance, the ligand may be conjugated to the sense strand, in particular, the 3′-end of the sense strand.

iRNA Conjugates

The iRNA agents disclosed herein can be in the form of conjugates. The conjugate may be attached at any suitable location in the iRNA molecule, e.g., at the 3′ end or the 5′ end of the sense or the antisense strand. The conjugates are optionally attached via a linker.

In some embodiments, an iRNA agent described herein is chemically linked to one or more ligands, moieties or conjugates, which may confer functionality, e.g., by affecting (e.g., enhancing) the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In some embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In some embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Typical ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Examples of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an a helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a neuron. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present disclosure as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the disclosure may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present disclosure may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present disclosure, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present disclosure are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipophilic Moieties

In certain embodiments, the lipophilic moiety is an aliphatic, cyclic such as alicyclic, or polycyclic such as polyalicyclic compound, such as a steroid (e.g., sterol) or a linear or branched aliphatic hydrocarbon. The lipophilic moiety may generally comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may comprise various substituents or one or more heteroatoms, such as an oxygen or nitrogen atom. Such lipophilic aliphatic moieties include, without limitation, saturated or unsaturated C₄-C₃₀ hydrocarbon (e.g., C₆-C₁₈ hydrocarbon), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C₁₀ terpenes, C₁₅ sesquiterpenes, C₂₀ diterpenes, C₃₀ triterpenes, and C₄₀ tetraterpenes), and other polyalicyclic hydrocarbons. For instance, the lipophilic moiety may contain a C₄-C₃₀ hydrocarbon chain (e.g., C₄-C₃₀ alkyl or alkenyl). In some embodiments the lipophilic moiety contains a saturated or unsaturated C₆-C₁₈ hydrocarbon chain (e.g., a linear C₆-C₁₈ alkyl or alkenyl). In some embodiments, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain (e.g., a linear C16 alkyl or alkenyl).

The lipophilic moiety may be attached to the RNAi agent by any method known in the art, including via a functional grouping already present in the lipophilic moiety or introduced into the RNAi agent, such as a hydroxy group (e.g., —CO—CH₂-OH). The functional groups already present in the lipophilic moiety or introduced into the RNAi agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

Conjugation of the RNAi agent and the lipophilic moiety may occur, for example, through formation of an ether or a carboxylic or carbamoyl ester linkage between the hydroxy and an alkyl group R—, an alkanoyl group RCO— or a substituted carbamoyl group RNHCO—. The alkyl group R may be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chained or branched; and saturated or unsaturated). Alkyl group R may be a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl group, or the like.

In some embodiments, the lipophilic moiety is conjugated to the double-stranded RNAi agent via a linker a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.

In other embodiments, the lipophilic moiety is a steroid, such as sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentanophenanthrene ring system. Steroids include, without limitation, bile acids (e.g., cholic acid, deoxycholic acid and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids, such as cortisone. A “cholesterol derivative” refers to a compound derived from cholesterol, for example by substitution, addition or removal of substituents.

In other embodiments, the lipophilic moiety is an aromatic moiety. In this context, the term “aromatic” refers broadly to mono- and polyaromatic hydrocarbons. Aromatic groups include, without limitation, C₆-C₁₄ aryl moieties comprising one to three aromatic rings, which may be optionally substituted; “aralkyl” or “arylalkyl” groups comprising an aryl group covalently linked to an alkyl group, either of which may independently be optionally substituted or unsubstituted; and “heteroaryl” groups. As used herein, the term “heteroaryl” refers to groups having 5 to 14 ring atoms, e.g., 5, 6, 9, or 10 ring atoms; having 6, 10, or 14π electrons shared in a cyclic array, and having, in addition to carbon atoms, one to about three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

As employed herein, a “substituted” alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group is one having one to about four, one to about three, or one or two, non-hydrogen substituents. Suitable substituents include, without limitation, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.

In some embodiments, the lipophilic moiety is an aralkyl group, e.g., a 2-arylpropanoyl moiety. The structural features of the aralkyl group are selected so that the lipophilic moiety will bind to at least one protein in vivo. In certain embodiments, the structural features of the aralkyl group are selected so that the lipophilic moiety binds to serum, vascular, or cellular proteins. In certain embodiments, the structural features of the aralkyl group promote binding to albumin, an immunoglobulin, a lipoprotein, α-2-macroglubulin, or α-1-glycoprotein.

In certain embodiments, the ligand is naproxen or a structural derivative of naproxen. Procedures for the synthesis of naproxen can be found in U.S. Pat. Nos. 3,904,682 and 4,009,197, which are hereby incorporated by reference in their entirety. Naproxen has the chemical name (S)-6-Methoxy-α-methyl-2-naphthaleneacetic acid and the structure is

In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. Procedures for the synthesis of ibuprofen can be found in U.S. Pat. No. 3,228,831, which is incorporated herein by reference for the methods provided therein. The structure of ibuprofen is

Additional exemplary aralkyl groups are illustrated in U.S. Pat. No. 7,626,014, which is incorporated herein by reference for the methods provided therein.

In other embodiments, suitable lipophilic moieties include lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

In certain embodiments, more than one lipophilic moiety can be incorporated into the double-strand RNAi agent, particularly when the lipophilic moiety has a low lipophilicity or hydrophobicity. In some embodiments, two or more lipophilic moieties are incorporated into the same strand of the double-strand RNAi agent. In some embodiments, each strand of the double-strand RNAi agent has one or more lipophilic moieties incorporated. In some embodiments, two or more lipophilic moieties are incorporated into the same position (i.e., the same nucleobase, same sugar moiety, or same internucleosidic linkage) of the double-strand RNAi agent. This can be achieved by, e.g., conjugating the two or more lipophilic moieties via a carrier, or conjugating the two or more lipophilic moieties via a branched linker, or conjugating the two or more lipophilic moieties via one or more linkers, with one or more linkers linking the lipophilic moieties consecutively.

The lipophilic moiety may be conjugated to the RNAi agent via a direct attachment to the ribosugar of the RNAi agent. Alternatively, the lipophilic moiety may be conjugated to the double-strand RNAi agent via a linker or a carrier.

In certain embodiments, the lipophilic moiety may be conjugated to the RNAi agent via one or more linkers (tethers).

In some embodiments, the lipophilic moiety is conjugated to the double-stranded RNAi agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.

B. Lipid Conjugates

In some embodiments, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can typically bind a serum protein, such as human serum albumin (HSA). An HSA binding ligand allows for vascular distribution of the conjugate to a target tissue. For example, the target tissue can be the central nervous system (CNS), e.g., brain and/or the spine, e.g., the dorsal root ganglion. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used.

A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid-based ligand can be used to modulate, e.g., control (e.g., inhibit) the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In some embodiments, the lipid-based ligand binds HSA. For example, the ligand can bind HSA with a sufficient affinity such that distribution of the conjugate to a non-kidney tissue is enhanced. However, the affinity is typically not so strong that the HSA-ligand binding cannot be reversed.

In some embodiments, the lipid-based ligand binds HSA weakly or not at all, such that distribution of the conjugate to the kidney is enhanced. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid-based ligand.

In other embodiments, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

Cell Permeation Agents

In other embodiments, the ligand is a cell-permeation agent, such as a helical cell-permeation agent. In some embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is typically an α-helical agent, and can have a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 3699). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 3700)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:3701)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 3702)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Typically, the peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the disclosure may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidomimetics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. In some embodiments, conjugates of this ligand target PECAM-1 or VEGF.

An RGD peptide moiety can be used to target a particular cell type, e.g., a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an dsRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing α_(V)β₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

Carbohydrate Conjugates and Ligands

In some embodiments of the compositions and methods of the disclosure, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, the compositions and methods of the disclosure include a C16 ligand. In exemplary embodiments, the C16 ligand of the disclosure has the following structure (exemplified here below for a uracil base, yet attachment of the C16 ligand is contemplated for a nucleotide presenting any base (C, G, A, etc.) or possessing any other modification as presented herein, provided that 2′ ribo attachment is preserved) and is attached at the 2′ position of the ribo within a residue that is so modified:

As shown above, a C16 ligand-modified residue presents a straight chain alkyl at the 2′-ribo position of an exemplary residue (here, a Uracil) that is so modified.

In exemplary embodiments, the C16 ligand of the disclosure can be conjugated to a ribonucleotide residue according to the following structure: possessing any other modification as presented herein, provided that 2′-ribo attachment is preserved) and is attached at the 2′-position of the ribo within a residue that is so modified:

where * denotes a bond to an adjacent nucleotide, and B is a nucleobase or a nucleobase analog, for example, where B is adenine, guanine, cytosine, thymine or uracil.

In some embodiments, a carbohydrate conjugate of a RNAi agent of the instant disclosure further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell permeation peptide.

Additional carbohydrate conjugates (and linkers) suitable for use in the present disclosure include those described in WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

In certain embodiments, the compositions and methods of the disclosure include a 5′-vinyl phosponate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, a 5′-vinyl phosphonate modified nucleotide of the disclosure has the structure of formula:

wherein X is O or S;

R is hydrogen, hydroxy, methoxy, fluoro, or C₁₋₂₀alkoxy (e.g., methoxy or n-hexadecyloxy);

R^(5′) is ═C(H)—P(O)(OH)₂ and the double bond between the C5′ carbon and R5′ is in the E or Z orientation (e.g., E orientation); and B is a nucleobase or a modified nucleobase, optionally where B is adenine, guanine, cytosine, thymine, or uracil. A vinyl phosponate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure. In certain embodiments, a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5′ end of the antisense strand of the dsRNA.

Vinyl phosphate modifications are also contemplated for the compositions and methods of the instant disclosure. An exemplary vinyl phosphate structure is:

for example, including the preceding structure where R^(5′) is ═C(H)—OP(O)(OH)₂ and the double bond between the C5′ carbon and R5′ is in the E or Z orientation (e.g., E orientation).

In some embodiments, a carbohydrate conjugate comprises a monosaccharide. In some embodiments, the monosaccharide is an N-acetylgalactosamine (GalNAc). GalNAc conjugates, which comprise one or more N-acetylgalactosamine (GalNAc) derivatives, are described, for example, in U.S. Pat. No. 8,106,022, the entire content of which is hereby incorporated herein by reference. In some embodiments, the GalNAc conjugate serves as a ligand that targets the iRNA to particular cells. In some embodiments, the GalNAc conjugate targets the iRNA to liver cells, e.g., by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be attached via a linker, e.g., a bivalent or trivalent branched linker. In some embodiments the GalNAc conjugate is conjugated to the 3′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3′ end of the sense strand) via a linker, e.g., a linker as described herein.

In some embodiments, the GalNAc conjugate is

In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic, wherein X is O or S:

In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below:

In some embodiments, a carbohydrate conjugate for use in the compositions and methods of the disclosure is selected from the group consisting of:

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

In some embodiments, an iRNA of the disclosure is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the disclosure include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

E. Thermally Destabilizing Modifications

In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating thermally destabilizing modifications in the seed region of the antisense strand (i.e., at positions 2-9 of the 5′-end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been discovered that dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5′ end, of the antisense strand have reduced off-target gene silencing activity. Accordingly, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five, or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region of the antisense strand. In some embodiments, one or more thermally destabilizing modification(s) of the duplex is/are located in positions 2-9, or positions 4-8, from the 5′-end of the antisense strand. In some further embodiments, the thermally destabilizing modification(s) of the duplex is/are located at position 6, 7, or 8 from the 5′-end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5′-end of the antisense strand. The term “thermally destabilizing modification(s)” includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) (e.g., a Tm with one, two, three, or four degrees lower than the Tm of the dsRNA without having such modification(s). In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5, or 9 from the 5′-end of the antisense strand.

The thermally destabilizing modifications can include, but are not limited to, abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2′-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA).

Exemplified abasic modifications include, but are not limited to, the following:

Wherein R=H, Me, Et or OMe; R′=H, Me, Et or OMe; R″=H, Me, Et or OMe

wherein B is a modified or unmodified nucleobase.

Exemplified sugar modifications include, but are not limited to the following:

wherein B is a modified or unmodified nucleobase.

In some embodiments the thermally destabilizing modification of the duplex is selected from the group consisting of:

wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.

The term “acyclic nucleotide” refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., C1′-C2′, C2′-C3′, C3′-C4′, C4′-C4′, or C1′-C4′) is absent or at least one of ribose carbons or oxygen (e.g., C1′, C2′, C3′, C4′, or C4′) are independently or in combination absent from the nucleotide. In some embodiments, acyclic nucleotide is

wherein B is a modified or unmodified nucleobase, R¹ and R² independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomers with bonds between C1′-C4′ being removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar is removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety). The acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings. The acyclic nucleotide can be linked via 2′-5′ or 3′-5′ linkage.

The term ‘GNA’ refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds:

The thermally destabilizing modification of the duplex can be mismatches (i.e., noncomplementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Other mismatch base pairings known in the art are also amenable to the present invention. A mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides. In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2′-deoxy nucleobase; e.g., the 2′-deoxy nucleobase is in the sense strand.

In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes nucleotides with impaired W-C H-bonding to complementary base on the target mRNA, such as:

More examples of abasic nucleotide, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO 2011/133876, which is herein incorporated by reference in its entirety.

The thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.

In some embodiments, the thermally destabilizing modification of the duplex includes nucleotides with non-canonical bases such as, but not limited to, nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010/0011895, which is herein incorporated by reference in its entirety. Exemplary nucleobase modifications are:

In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more α-nucleotide complementary to the base on the target mRNA, such as:

wherein R is H, OH, OCH₃, F, NH₂, NHMe, NMe₂ or O-alkyl.

Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are:

The alkyl for the R group can be a C₁-C₆alkyl. Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.

As the skilled artisan will recognize, in view of the functional role of nucleobases is defining specificity of a RNAi agent of the disclosure, while nucleobase modifications can be performed in the various manners as described herein, e.g., to introduce destabilizing modifications into a RNAi agent of the disclosure, e.g., for purpose of enhancing on-target effect relative to off-target effect, the range of modifications available and, in general, present upon RNAi agents of the disclosure tends to be much greater for non-nucleobase modifications, e.g., modifications to sugar groups or phosphate backbones of polyribonucleotides. Such modifications are described in greater detail in other sections of the instant disclosure and are expressly contemplated for RNAi agents of the disclosure, either possessing native nucleobases or modified nucleobases as described above or elsewhere herein.

In addition to the antisense strand comprising a thermally destabilizing modification, the dsRNA can also comprise one or more stabilizing modifications. For example, the dsRNA can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitations, the stabilizing modifications all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two stabilizing modifications. The stabilizing modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the stabilizing modification can occur on every nucleotide on the sense strand or antisense strand; each stabilizing modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both stabilizing modification in an alternating pattern. The alternating pattern of the stabilizing modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the stabilizing modifications on the sense strand can have a shift relative to the alternating pattern of the stabilizing modifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitations, a stabilizing modification in the antisense strand can be present at any positions.

In some embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5′-end. In some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5′-end. In still some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14, and 16 from the 5′-end.

In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification can be the nucleotide at the 5′-end or the 3′-end of the destabilizing modification, i.e., at position −1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5′-end and the 3′-end of the destabilizing modification, i.e., positions −1 and +1 from the position of the destabilizing modification.

In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3′-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the sense strand can be present at any positions. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5′-end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5′-end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three, or four stabilizing modifications.

In some embodiments, the sense strand does not comprise a stabilizing modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.

Exemplary thermally stabilizing modifications include, but are not limited to, 2′-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

In some embodiments, the dsRNA of the disclosure comprises at least four (e.g., four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, the 2′-fluoro nucleotides all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two 2′-fluoro nucleotides. The 2′-fluoro modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the 2′-fluoro modification can occur on every nucleotide on the sense strand or antisense strand; each 2′-fluoro modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both 2′-fluoro modifications in an alternating pattern. The alternating pattern of the 2′-fluoro modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the 2′-fluoro modifications on the sense strand can have a shift relative to the alternating pattern of the 2′-fluoro modifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, a 2′-fluoro modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5′-end. In some other embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5′-end. In still some other embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 14, and 16 from the 5′-end.

In some embodiments, the antisense strand comprises at least one 2′-fluoro nucleotide adjacent to the destabilizing modification. For example, the 2′-fluoro nucleotide can be the nucleotide at the 5′-end or the 3′-end of the destabilizing modification, i.e., at position −1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a 2′-fluoro nucleotide at each of the 5′-end and the 3′-end of the destabilizing modification, i.e., positions −1 and +1 from the position of the destabilizing modification.

In some embodiments, the antisense strand comprises at least two 2′-fluoro nucleotides at the 3′-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, a 2′-fluoro modification in the sense strand can be present at any positions. In some embodiments, the antisense comprises 2′-fluoro nucleotides at positions 7, 10, and 11 from the 5′-end. In some other embodiments, the sense strand comprises 2′-fluoro nucleotides at positions 7, 9, 10, and 11 from the 5′-end. In some embodiments, the sense strand comprises 2′-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some other embodiments, the sense strand comprises 2′-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three, or four 2′-fluoro nucleotides.

In some embodiments, the sense strand does not comprise a 2′-fluoro nucleotide in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.

In some embodiments, the dsRNA molecule of the disclosure comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the antisense strand contains at least one thermally destabilizing nucleotide, where the at least one thermally destabilizing nucleotide occurs in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), wherein one end of the dsRNA is blunt, while the other end is comprises a 2 nt overhang, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA comprises a blunt end at 5′-end of the antisense strand. In some embodiments, the 2 nt overhang is at the 3′-end of the antisense.

In some embodiments, every nucleotide in the sense strand and antisense strand of the dsRNA molecule may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, or 2′-fluoro. The strands can contain more than one modification. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. It is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-deoxy, 2′-O-methyl, or 2′-fluoro modifications, acyclic nucleotides or others. In some embodiments, the sense strand and antisense strand each comprises two differently modified nucleotides selected from 2′-O-methyl or 2′-deoxy. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl nucleotide, 2′-deoxy nucleotide, 2′-deoxy-2′-fluoro nucleotide, 2′-O-N-methylacetamido (2′-O-NMA) nucleotide, a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, 2′-O-aminopropyl (2′-O-AP) nucleotide, or 2′-ara-F nucleotide. Again, it is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand.

In some embodiments, the dsRNA molecule of the disclosure comprises modifications of an alternating pattern, particular in the B1, B2, B3, B1′, B2′, B3′, B4′ regions. The term “alternating motif” or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In some embodiments, the dsRNA molecule of the disclosure comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 3′-5′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 3′-5′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

The dsRNA molecule of the disclosure may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.

In some embodiments, the dsRNA molecule comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. In some embodiments, these terminal three nucleotides may be at the 3′-end of the antisense strand.

In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, or 4 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within positions 1-10 of the termini position(s) of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage at one end or both ends of the sense or antisense strand.

In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within positions 1-10 of the internal region of the duplex of each of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate methylphosphonate internucleotide linkage at position 8-16 of the duplex region counting from the 5′-end of the sense strand; the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modification within positions 1-10 of the termini position(s).

In some embodiments, the dsRNA molecule of the disclosure further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 18-23 of the sense strand (counting from the 5′-end), and one to five phosphorothioate or methylphosphonate internucleotide linkage modification at positions 1 and 2 and one to five within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 (counting from the 5′-end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 (counting from the 5′-end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 20 and 21 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 21 and 22 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 22 and 23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 the antisense strand (counting from the 5′-end).

In some embodiments, compound of the disclosure comprises a pattern of backbone chiral centers. In some embodiments, a common pattern of backbone chiral centers comprises at least 5 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 6 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 9 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 19 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration, and no more than 8 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration, and no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration, and no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration, and no more than 4 internucleotidic linkages which are not chiral. In some embodiments, the internucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages which are not chiral are optionally contiguous or not contiguous.

In some embodiments, compound of the disclosure comprises a block is a stereochemistry block. In some embodiments, a block is an Rp block in that each internucleotidic linkage of the block is Rp. In some embodiments, a 5′-block is an Rp block. In some embodiments, a 3′-block is an Rp block. In some embodiments, a block is an Sp block in that each internucleotidic linkage of the block is Sp. In some embodiments, a 5′-block is an Sp block. In some embodiments, a 3′-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each internucleotidic linkage in a natural phosphate linkage.

In some embodiments, compound of the disclosure comprises a 5′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block comprises 4 or more nucleoside units. In some embodiments, a 5′-block comprises 5 or more nucleoside units. In some embodiments, a 5′-block comprises 6 or more nucleoside units. In some embodiments, a 5′-block comprises 7 or more nucleoside units. In some embodiments, a 3′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block comprises 4 or more nucleoside units. In some embodiments, a 3′-block comprises 5 or more nucleoside units. In some embodiments, a 3′-block comprises 6 or more nucleoside units. In some embodiments, a 3′-block comprises 7 or more nucleoside units.

In some embodiments, compound of the disclosure comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of internucleotidic linkage, e.g., natural phosphate linkage, modified internucleotidic linkage, Rp chiral internucleotidic linkage, Sp chiral internucleotidic linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

In some embodiments, the dsRNA molecule of the disclosure comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In some embodiments, the dsRNA molecule of the disclosure comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In some embodiments, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

It was found that introducing 4′-modified or 5′-modified nucleotide to the 3′-end of a phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage of a dinucleotide at any position of single stranded or double stranded oligonucleotide can exert steric effect to the internucleotide linkage and, hence, protecting or stabilizing it against nucleases.

In some embodiments, 5′-modified nucleoside is introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. For instance, a 5′-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The alkyl group at the 5′ position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 4′-modified nucleoside is introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. For instance, a 4′-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The alkyl group at the 4′ position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemic or chirally pure R or S isomer. Alternatively, a 4′-O-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The 4′-O-alkyl of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 5′-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 4′-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 4′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 4′-O-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, the dsRNA molecule of the disclosure can comprise 2′-5′ linkages (with 2′-H, 2′-OH, and 2′-OMe and with P═O or P═S). For example, the 2′-5′ linkages modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.

In other embodiments, the dsRNA molecule of the disclosure can comprise L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe). For example, these L sugars modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.

Various publications describe multimeric siRNA which can all be used with the dsRNA of the disclosure. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520 which are hereby incorporated by their entirely.

In some embodiments dsRNA molecules of the disclosure are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)₂(O)P—O-5′); 5′-diphosphate ((HO)₂(O)P—O-P(HO)(O)-O-5′); 5′-triphosphate ((HO)₂(O)P—O-(HO)(O)P—O—P(HO)(O)-O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P-O—(HO)(O)P—O-P(HO)(O)-O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5′-(HO)(O)P-O—(HO)(O)P—O-P(HO)(O)-O-5′); 5′-monothiophosphate (phosphorothioate; (HO)₂(S)P-O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)₂(O)P-S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P-NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)-O-5′-, 5′-alkenylphosphonates (i.e. vinyl, substituted vinyl), (OH)₂(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)-O-5′-). In one example, the modification can in placed in the antisense strand of a dsRNA molecule.

Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In some embodiments, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

In some embodiments, a dsRNA of the disclosure is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI)-(XXXIV):

wherein:

-   -   q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent         independently for each occurrence 0-20 and wherein the repeating         unit can be the same or different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C) are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C) are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), CEC or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C) are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N-O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) and L^(5C) represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):

wherein L⁵, L^(5B) and L^(5C) represent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a some embodiments, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a suitable pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In some embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

Redox Cleavable Linking Groups

In some embodiments, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

In some embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O-P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—, wherein Rk at each occurrence can be, independently, C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. In some embodiments, phosphate-based linking groups are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. In some embodiments, a phosphate-based linking group is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

Acid Cleavable Linking Groups

In some embodiments, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In some embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). In some embodiments, the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

Ester-Based Cleavable Linking Groups

In some embodiments, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

Peptide-Based Cleavable Linking Groups

In some embodiments, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide-based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above. Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an iRNA. The present disclosure also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds, or “chimeras,” in the context of the present disclosure, are iRNA compounds, e.g., dsRNAs, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

Delivery of iRNA

The delivery of an iRNA to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising an iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

Direct Delivery

In general, any method of delivering a nucleic acid molecule can be adapted for use with an iRNA (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver an iRNA molecule in vivo: (1) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, the spine) or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo.

Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to other groups, e.g., a lipid or carbohydrate group as described herein. Such conjugates can be used to target iRNA to particular cells, e.g., liver cells, e.g., hepatocytes. For example, GalNAc conjugates or lipid (e.g., LNP) formulations can be used to target iRNA to particular cells, e.g., liver cells, e.g., hepatocytes.

iRNA molecules can also be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

Vector Encoded iRNAs

In some embodiments, iRNA targeting SCN9A can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively, each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In some embodiments, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

An iRNA expression vector is typically a DNA plasmid or viral vector. An expression vector compatible with eukaryotic cells, e.g., with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors contain convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

An iRNA expression plasmid can be transfected into a target cell as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the disclosure. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.

Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.

In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the disclosure, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In some embodiments, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the disclosure, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol., 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

Another typical viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

III. Pharmaceutical Compositions Containing iRNA

In some embodiments, the disclosure provides pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the iRNA is useful for treating a disease or disorder related to the expression or activity of SCN9A (e.g., pain, e.g., chronic pain or pain-related disorder). Such pharmaceutical compositions are formulated based on the mode of delivery. In some embodiments, compositions can be formulated for localized delivery, e.g., by CNS delivery (e.g., intrathecal, intracranial, intracerebral, intraventricular, epidural, or intraganglionic routes of injection, optionally by infusion into the brain or spine, e.g., by continuous pump infusion). In another example, compositions can be formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery, intramuscular (IM), or subcutaneous delivery (subQ). In some embodiments, a composition provided herein (e.g., a composition comprising a GalNAc conjugate or an LNP formulation) is formulated for intravenous delivery.

The pharmaceutical compositions featured herein are administered in a dosage sufficient to inhibit expression of SCN9A. In general, a suitable dose of iRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.

In some embodiments, a repeat-dose regimen may include administration of a therapeutic amount of a RNAi agent on a regular basis, such as monthly to once every six months. In certain embodiments, the RNAi agent is administered about once per quarter (i.e., about once every three months) to about twice per year.

After an initial treatment regimen (e.g., loading dose), the treatments can be administered on a less frequent basis.

In other embodiments, the pharmaceutical composition may be administered once daily, or the iRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as can be used with the agents of the present disclosure. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

The effect of a single dose on SCN9A levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5-day intervals, or at not more than 1, 2, 3, 4, 12, 24, or 36-week intervals.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the disclosure can be made using conventional methodologies or on the basis of in vivo testing using a suitable animal model.

A suitable animal model, e.g., a mouse or a cynomolgus monkey, e.g., an animal containing a transgene expressing human SCN9A, can be used to determine the therapeutically effective dose and/or an effective dosage regimen administration of SCN9A siRNA.

In some embodiments, the iRNA compounds described herein can be delivered in a manner to target a particular tissue, such as the CNS (e.g., optionally the brain or spine tissue, e.g., cortex, cerebellum, dorsal root ganglia, substantia nigra, cerebellar dentate nucleus, pallidum, striatum, brainstem, thalamus, subthalamic, red, and pontine nuclei, cranial nerve nuclei and the anterior horn; and Clarke's column of the spinal cord cervical spine, lumbar spine, or thoracic spine).

The present disclosure also includes pharmaceutical compositions and formulations that include the iRNA compounds featured herein. The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be local (e.g., by intrathecal, intraventricular, intracranial, epidural, or intraganglionic injection), topical (e.g., buccal and sublingual administration), oral, intravitreal, transdermal, airway (aerosol), nasal, rectal, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal, or intraventricular administration.

In some embodiments, the administration is via a bolus injection. In some embodiments, the administration is via a depot injection. A depot injection may release the RNAi agent in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of SCN9A, or a therapeutic or prophylactic effect.

In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intracranial, intravenous, or epidural infusions. In certain embodiments, the pump is a surgically implanted pump that delivers the RNAi agent to the CNS.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the iRNAs featured in the disclosure are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the disclosure may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present disclosure, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C_(1215G), that contains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.). Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In some embodiments, an SCN9A dsRNA featured in the disclosure is fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present disclosure typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present disclosure are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In some embodiments, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In some embodiments, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

In some embodiments, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

In some embodiments, the iRNA is formulated in a lipid nanoparticle (LNP).

LNP01

In some embodiments, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (e.g., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are provided in the following table.

TABLE 7 Exemplary lipid formulations cationic lipid/non-cationic lipid/ cholesterol/PEG-lipid conjugate Cationic Lipid Lipid:siRNA ratio SNALP 1,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-cDMA dimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA ~7:1 S-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DPPC/Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA ~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH- 50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100) Lipid:siRNA 10:1 LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl 4-(dimethylamino)butanoate 50/10/38.5/1.5 (MC3) Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- C12-200/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:siRNA 10:1 yl)ethylazanediyl)didodecan-2-ol (C12-200) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG- DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000) SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference. XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. 61/185,712, filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference. MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and International Application No. PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated by reference. ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference. C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

Synthesis of Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles featured in the disclosure may be prepared by known organic synthesis techniques. All substituents are as defined below unless indicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y), wherein n is 0, 1 or 2, R^(x) and R^(y) are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y).

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods featured in the disclosure may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this disclosure are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles featured in the disclosure are formulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.

Lipid A, where R₁ and R₂ are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R₃ and R₄ are independently lower alkyl or R₃ and R₄ can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:

Synthesis of 515:

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516:

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO₃ solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H] −232.3 (96.94%).

Synthesis of 517A and 517B:

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO₃(1×50 mL) solution, water (1×30 mL) and finally with brine (1×50 mL). Organic phase was dried over an.Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS-[M+H] −266.3, [M+NH4+] −283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518:

Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.

Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the disclosure are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the disclosure may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intravitreal, intraventricular, or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations featured in the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions featured in the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Additional Formulations

Emulsions

The compositions of the present disclosure may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise, a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In some embodiments of the present disclosure, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically, microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotopically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present disclosure will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

Microemulsions of the present disclosure may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present disclosure. Penetration enhancers used in the microemulsions of the present disclosure may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Penetration Enhancers

In some embodiments, the present disclosure employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above-mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present disclosure, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with the present disclosure, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present disclosure, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of β-diketones (enamines) (see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invivogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

Certain compositions of the present disclosure also incorporate carrier compounds in the formulation. As used herein, “carrier compound” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a pharmaceutical carrier or excipient may comprise, e.g., a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions, e.g., at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the disclosure include (a) one or more iRNA compounds and (b) one or more biologic agents which function by a non-RNAi mechanism. Examples of such biologic agents include agents that interfere with an interaction of SCN9A and at least one SCN9A binding partner.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are typical.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the disclosure lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the disclosure can be administered in combination with other known agents effective in treatment of diseases or disorders related to SCN9A expression (e.g., pain, e.g., chronic pain or pain-related disorder). In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Methods of Treating Disorders Related to Expression of SCN9A

The present disclosure relates to the use of an iRNA targeting SCN9A to inhibit SCN9A expression and/or to treat a disease, disorder, or pathological process that is related to SCN9A expression (e.g., pain, e.g., chronic pain or pain-related disorder).

In some aspects, a method of treatment of a disorder related to expression of SCN9A is provided, the method comprising administering an iRNA (e.g., a dsRNA) disclosed herein to a subject in need thereof. In some embodiments, the iRNA inhibits (decreases) SCN9A expression.

In some embodiments, the subject is an animal that serves as a model for a disorder related to SCN9A expression, e.g., pain, e.g., chronic pain or pain-related disorder, e.g., inflammatory pain, neuropathic pain, pain hypersensitivity, pain hyposensitivity, inability to sense pain, primary erythromelalgia (PE), paroxysmal extreme pain disorder (PEPD), small fiber neuropathy (SFN), trigeminal neuralgia (TN), and pain associated with, e.g., cancer, arthritis, diabetes, traumatic injury and viral infections.

Chronic Pain and Pain-Related Disorders

In some embodiments, the disorder related to SCN9A expression is pain, e.g., chronic pain or pain related disorders, e.g., pain hypersensitivity or hyposensitivity. Non-limiting examples of pain-related disorders that are treatable using the methods described herein include inflammatory pain, neuropathic pain, pain insensitivity, primary erythromelalgia (PE), paroxysmal extreme pain disorder (PEPD), small fiber neuropathy (SFN), trigeminal neuralgia (TN), and pain associated with cancer, arthritis, diabetes, traumatic injury, and viral infections. In some embodiments, the pain-related disorder is an inherited pain-related disorder, e.g., PE and PEPD.

Clinical and pathological features of pain-related disorders include, but are not limited to, burning pain, redness of skin, flushing, warmth of extremities, joint pain, severe pain, e.g., periods of severe pain in the lower body, upper body (e.g., pain in the eyes or jaw), or extremities (e.g., hands and feet), inability to sense pain, fatigue, and/or insomnia.

In some embodiments, the subject with the pain, e.g., chronic pain, or pain-related disorder is less than 18 years old. In some embodiments, the subject with the pain, e.g., chronic pain, or pain-related disorder is an adult. In some embodiments, the subject has, or is identified as having, elevated levels of SCN9A mRNA or protein relative to a reference level (e.g., a level of SCN9A that is greater than a reference level).

In some embodiments, the pain, e.g., chronic pain, or the pain-related disorder is diagnosed using analysis of a sample from the subject (e.g., an aqueous cerebral spinal fluid (CSF) sample). In some embodiments, the sample is analyzed using a method selected from one or more of: fluorescent in situ hybridization (FISH), immunohistochemistry, SCN9A immunoassay, electron microscopy, laser microdissection, and mass spectrometry. In some embodiments, pain, e.g., chronic pain, or pain-related disorder is diagnosed using any suitable diagnostic test or technique, e.g., SCN9A mutation testing, a measure of pain sensitivity, a measure of pain threshold, a measure of pain level, and/or a measure of pain disability level (Dansie and Turk 2013 Br J Anaesth 111(1):19-25).

Combination Therapies

In some embodiments, an iRNA (e.g., a dsRNA) disclosed herein is administered in combination with a second therapy (e.g., one or more additional therapies) known to be effective in treating a disorder related to SCN9A expression (e.g., pain, e.g., chronic pain or pain-related disorder) or a symptom of such a disorder. The iRNA may be administered before, after, or concurrent with the second therapy. In some embodiments, the iRNA is administered before the second therapy. In some embodiments, the iRNA is administered after the second therapy. In some embodiments, the iRNA is administered concurrent with the second therapy.

The second therapy may be an additional therapeutic agent. The iRNA and the additional therapeutic agent can be administered in combination in the same composition or the additional therapeutic agent can be administered as part of a separate composition.

In some embodiments, the second therapy is a non-iRNA therapeutic agent that is effective to treat the disorder or symptoms of the disorder.

In some embodiments, the iRNA is administered in conjunction with a therapy.

Exemplary combination therapies include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen, opioids, or corticosteroids, acupuncture, therapeutic massage, dorsal root ganglion stimulation, spinal cord stimulation, or topical pain relievers.

Administration Dosages, Routes, and Timing

A subject (e.g., a human subject, e.g., a patient) can be administered a therapeutic amount of iRNA. The therapeutic amount can be, e.g., 0.05-50 mg/kg. For example, the therapeutic amount can be 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, or 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/kg dsRNA.

In some embodiments, the iRNA is formulated for delivery to a target organ, e.g., to the brain or spinal chord.

In some embodiments, the iRNA is formulated as a lipid formulation, e.g., an LNP formulation as described herein. In some such embodiments, the therapeutic amount is 0.05-5 mg/kg, e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg/kg dsRNA. In some embodiments, the lipid formulation, e.g., LNP formulation, is administered intravenously. In some embodiments, the iRNA (e.g., dsRNA) is formulated as an LNP formulation and is administered (e.g., intravenously, intrathecally, intracerebrally, intracranially, or intraventricularly administered) at a dose of 0.1 to 1 mg/kg.

In some embodiments, the iRNA is administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

In some embodiments, the iRNA is in the form of a lipophilic conjugate (e.g., a C16 conjugate) as described herein. In some such embodiments, the therapeutic amount is 0.5-50 mg, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/kg dsRNA. In some embodiments, the lipophilic conjugate (e.g., a C16 conjugate) is administered subcutaneously. In some embodiments, the iRNA (e.g., dsRNA) is in the form of a lipophilic conjugate and is administered (e.g., subcutaneously administered) at a dose of 1 to 10 mg/kg. In some embodiments, the iRNA is in the form of a GalNAc conjugate e.g., as described herein. In some such embodiments, the therapeutic amount is 0.5-50 mg, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/kg dsRNA. In some embodiments, the e.g., GalNAc conjugate is administered subcutaneously.

In some embodiments, the administration is repeated, for example, on a regular basis, such as, daily, biweekly (i.e., every two weeks) for one month, two months, three months, four months, six months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer.

In some embodiments, the iRNA agent is administered in two or more doses. In some embodiments, the number or amount of subsequent doses is dependent on the achievement of a desired effect, e.g., to (a) reduce pain; (b) inhibit or reduce the expression or activity of SCN9A or the achievement of a therapeutic or prophylactic effect, e.g., reduction or prevention of one or more symptoms associated with the disorder.

In some embodiments, the iRNA agent is administered according to a schedule. For example, the iRNA agent may be administered once per week, twice per week, three times per week, four times per week, or five times per week. In some embodiments, the schedule involves regularly spaced administrations, e.g., hourly, every four hours, every six hours, every eight hours, every twelve hours, daily, every 2 days, every 3 days, every 4 days, every 5 days, weekly, biweekly, or monthly. In some embodiments, the iRNA agent is administered at the frequency required to achieve a desired effect.

In some embodiments, the schedule involves closely spaced administrations followed by a longer period of time during which the agent is not administered. For example, the schedule may involve an initial set of doses that are administered in a relatively short period of time (e.g., about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours) followed by a longer time period (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks) during which the iRNA agent is not administered. In some embodiments, the iRNA agent is initially administered hourly and is later administered at a longer interval (e.g., daily, weekly, biweekly, or monthly). In some embodiments, the iRNA agent is initially administered daily and is later administered at a longer interval (e.g., weekly, biweekly, or monthly). In certain embodiments, the longer interval increases over time or is determined based on the achievement of a desired effect.

Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion dose, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted effects.

Methods for Modulating Expression of SCN9A

In some aspects, the disclosure provides a method for modulating (e.g., inhibiting or activating) the expression of SCN9A, e.g., in a cell, in a tissue, or in a subject. In some embodiments, the cell or tissue is ex vivo, in vitro, or in vivo. In some embodiments, the cell or tissue is in the central nervous system (e.g., brain or spine tissue, e.g., cortex, cerebellum, dorsal root ganglia, substantia nigra, cerebellar dentate nucleus, pallidum, striatum, brainstem, thalamus, subthalamic, red, and pontine nuclei, cranial nerve nuclei and the anterior horn; and Clarke's column of the spinal cord cervical spine, lumbar spine, or thoracic spine). In some embodiments, the cell or tissue is in a subject (e.g., a mammal, such as, for example, a human) In some embodiments, the subject (e.g., the human) is at risk, or is diagnosed with a disorder related to expression of SCN9A expression, as described herein.

In some embodiments, the method includes contacting the cell with an iRNA as described herein, in an amount effective to decrease the expression of SCN9A in the cell. In some embodiments, contacting a cell with an RNAi agent includes contacting a cell in vitro with the RNAi agent or contacting a cell in vivo with the RNAi agent. In some embodiments, the RNAi agent is put into physical contact with the cell by the individual performing the method, or the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell. Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., a CNS tissue. For example, the RNAi agent may contain or be coupled to a ligand, e.g., a lipophilic moiety or moieties as described below and further detailed, e.g., in PCT/US2019/031170 which is incorporated herein by reference in its entirety, including the passages therein describing lipophilic moieties, that directs or otherwise stabilizes the RNAi agent at a site of interest. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.

The expression of SCN9A may be assessed based on the level of expression of SCN9A mRNA, SCN9A protein, or the level of another parameter functionally linked to the level of expression of SCN9A. In some embodiments, the expression of SCN9A is inhibited by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the iRNA has an IC₅₀ in the range of 0.001-0.01 nM, 0.001-0.10 nM, 0.001-1.0 nM, 0.001-10 nM, 0.01-0.05 nM, 0.01-0.50 nM, 0.02-0.60 nM, 0.01-1.0 nM, 0.01-1.5 nM, 0.01-10 nM. The IC₅₀ value may be normalized relative to an appropriate control value, e.g., the IC₅₀ of a non-targeting iRNA.

In some embodiments, the method includes introducing into the cell or tissue an iRNA as described herein and maintaining the cell or tissue for a time sufficient to obtain degradation of the mRNA transcript of SCN9A, thereby inhibiting the expression of SCN9A in the cell or tissue.

In some embodiments, the method includes administering a composition described herein, e.g., a composition comprising an iRNA that binds SCN9A, to the mammal such that expression of the target SCN9A is decreased, such as for an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, or four weeks or longer. In some embodiments, the decrease in expression of SCN9A is detectable within 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours of the first administration.

In some embodiments, the method includes administering a composition as described herein to a mammal such that expression of the target SCN9A is increased by e.g., at least 10% compared to an untreated animal. In some embodiments, the activation of SCN9A occurs over an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, four weeks, or more. Without wishing to be bound by theory, an iRNA can activate SCN9A expression by stabilizing the SCN9A mRNA transcript, interacting with a promoter in the genome, or inhibiting an inhibitor of SCN9A expression.

The iRNAs useful for the methods and compositions featured in the disclosure specifically target RNAs (primary or processed) of SCN9A. Compositions and methods for inhibiting the expression of SCN9A using iRNAs can be prepared and performed as described elsewhere herein.

In some embodiments, the method includes administering a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of SCN9A of the subject, e.g., the mammal, e.g., the human, to be treated. The composition may be administered by any appropriate means known in the art including, but not limited to intracranial, intrathecal, intraventricular, topical, and intravenous administration.

In certain embodiments, the composition is administered, e.g., using oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, intracranial, and intrathecal), intravenous, intramuscular, intravitreal, subcutaneous, transdermal, airway (aerosol), nasal, or rectal. In other embodiments, the composition is administered topically (e.g., buccal and sublingual administration). In other embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by intrathecal injection. In certain embodiments, the compositions are administered by intraventricular injection. In certain embodiments, the compositions are administered by intracranial injection. In certain embodiments, the compositions are administered by epidural injection. In certain embodiments, the compositions are administered by intraganglionic injection.

In certain embodiments, the composition is administered by intravenous infusion or injection. In some such embodiments, the composition comprises a lipid formulated siRNA (e.g., an LNP formulation, such as an LNP11 formulation) for intravenous infusion.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Specific Embodiments

1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression sodium channel, voltage gated, type IX alpha subunit (SCN9A), wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of a coding strand of human SCN9A and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of a non-coding strand of human SCN9A such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.

2. The dsRNA agent of embodiment 1, wherein the coding strand of human SCN9A comprises the sequence SEQ ID NO: 1.

3. The dsRNA agent of embodiment 1 or 2, wherein the non-coding strand of human SCN9A comprises the sequence of SEQ ID NO: 2.

4 The dsRNA agent of embodiment 1, wherein the coding strand of human SCN9A comprises the sequence SEQ ID NO: 4001.

5. The dsRNA agent of embodiment 1 or 4, wherein the non-coding strand of human SCN9A comprises the sequence of SEQ ID NO: 4002.

6. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of SCN9A, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.

7. The dsRNA agent of embodiment 6, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

8. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of SCN9A, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 4002 such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.

9. The dsRNA agent of embodiment 8, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 4001.

10. The dsRNA of any of the preceding embodiments, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 17 contiguous nucleotides in the antisense strand.

11. The dsRNA of embodiment 10, wherein the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

12. The dsRNA of any of the preceding embodiments, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 4002 such that the sense strand is complementary to the at least 17 contiguous nucleotides in the antisense strand.

13. The dsRNA of embodiment 12, wherein the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 4001.

14. The dsRNA of any of the preceding embodiments, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 19 contiguous nucleotides in the antisense strand.

15. The dsRNA of embodiment 14, wherein the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

16. The dsRNA of any of the preceding embodiments, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 4002 such that the sense strand is complementary to the at least 19 contiguous nucleotides in the antisense strand.

17. The dsRNA of embodiment 16, wherein the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 4001.

18. The dsRNA of any of the preceding embodiments, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 21 contiguous nucleotides in the antisense strand.

19. The dsRNA of embodiment 18, wherein the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

20. The dsRNA of any of the preceding embodiments, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 4002 such that the sense strand is complementary to the at least 21 contiguous nucleotides in the antisense strand.

21. The dsRNA of embodiment 20, wherein the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 4001.

22. The dsRNA agent of any one of embodiments 1-21, wherein the portion of the sense strand is a portion within nucleotides 581-601, 760-780, or 8498-8518 of SEQ ID NO: 4001.

23. The dsRNA agent of any one of embodiments 1-22, wherein the portion of the sense strand is a portion within a sense strand from a duplex chosen from AD-1251284 (UGUCGAGUACACUUUUACUGA (SEQ ID NO:4827)), AD-961334 (CAACACAATUTCUUCUUAGCA (SEQ ID NO: 5026)), or AD-1251325 (AAAACAAUCUUCCGUUUCAAA (SEQ ID NO: 4822)).

24. The dsRNA agent of any one of embodiments 1-23, wherein the portion of the sense strand is a sense strand chosen from the sense strands of AD-1251284 (UGUCGAGUACACUUUUACUGA (SEQ ID NO:4827)), AD-961334 (CAACACAATUTCUUCUUAGCA (SEQ ID NO: 5026)), or AD-1251325 (AAAACAAUCUUCCGUUUCAAA (SEQ ID NO: 4822)).

25. The dsRNA of any one of embodiments 1-24, wherein the portion of the antisense strand is a portion within an antisense strand from a duplex chosen from AD-1251284 (UCAGTAAAAGUGUACTCGACAUU (SEQ ID NO: 5093)), AD-961334 (UGCUAAGAAGAAATUGUGUUGUU (SEQ ID NO: 5292)), or AD-1251325 (UUUGAAACGGAAGAUUGUUUUCC (SEQ ID NO: 5088)).

26. The dsRNA of any one of embodiments 1-25, wherein the portion of the antisense strand is an antisense strand chosen the antisense strands of AD-1251284 (UCAGTAAAAGUGUACTCGACAUU (SEQ ID NO: 5093)), AD-961334 (UGCUAAGAAGAAATUGUGUUGUU (SEQ ID NO: 5292)), or AD-1251325 (UUUGAAACGGAAGAUUGUUUUCC (SEQ ID NO: 5088)).

27. The dsRNA of any one of embodiments 1-26, wherein the sense strand and the antisense strand comprise nucleotide sequences of the paired sense strand and antisense strand of a duplex selected from AD-1251284 (SEQ ID NO: 4827 and 5093), AD-961334 (SEQ ID NO: 5026 and 5292), or AD-1251325 (SEQ ID NO: 4822 and 5088).

28. The dsRNA agent of any one of the preceding embodiments, wherein the portion of the sense strand is a portion within a sense strand in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20.

29. The dsRNA agent of any one of the preceding embodiments, wherein the portion of the antisense strand is a portion within an antisense strand in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20.

30. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20.

31. The dsRNA agent of any of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 that corresponds to the antisense sequence.

32. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20.

33. The dsRNA agent of any of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 that corresponds to the antisense sequence.

34. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0,1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20.

35. The dsRNA agent of any of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 that corresponds to the antisense sequence.

36. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0,1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20.

37. The dsRNA agent of any of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 that corresponds to the antisense sequence.

38. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of SCN9A, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20, and the sense strand comprises a nucleotide sequence of a sense sequence listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 that corresponds to the antisense sequence.

39. The dsRNA agent of embodiment 38, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in Table 5A, and the sense strand comprises a nucleotide sequence of a sense sequence listed in Table 5A that corresponds to the antisense sequence.

40. The dsRNA agent of embodiment 38, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in Table 13A, and the sense strand comprises a nucleotide sequence of a sense sequence listed in Table 13A that corresponds to the antisense sequence.

41. The dsRNA agent of embodiment 38, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in Table 14A, and the sense strand comprises a nucleotide sequence of a sense sequence listed in Table 14A that corresponds to the antisense sequence.

42. The dsRNA agent of embodiment 38, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in Table 15A, and the sense strand comprises a nucleotide sequence of a sense sequence listed in Table 15A that corresponds to the antisense sequence.

43. The dsRNA agent of embodiment 38, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in Table 16, and the sense strand comprises a nucleotide sequence of a sense sequence listed in Table 16 that corresponds to the antisense sequence.

44. The dsRNA agent of any one of embodiments 38, wherein the dsRNA agent is AD-1251284, AD-961334, AD-1251325, AD-1331352, AD-1209344, or AD-1331350.

45. The dsRNA of any one of embodiments 38-44, wherein:

(i) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 4029, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4295;

(ii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 4228, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4494;

(iii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 5339, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 5355;

(iv) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 5800, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 5801;

(v) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 5526, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 5681; or

(vi) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 5542, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 5697.

46. The dsRNA agent of any of the preceding embodiments, wherein the sense strand is at least 23 nucleotides in length, e.g., 23-30 nucleotides in length.

47. The dsRNA agent of any of the preceding embodiments, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties.

48. The dsRNA agent of embodiment 47, wherein the lipophilic moiety is conjugated to one or more positions in the double stranded region of the dsRNA agent.

49. The dsRNA agent of embodiment 47 or 48, wherein the lipophilic moiety is conjugated via a linker or carrier.

50. The dsRNA agent of any one of embodiments 47-49, wherein lipophilicity of the lipophilic moiety, measured by logKow, exceeds 0.

51. The dsRNA agent of any one of the preceding embodiments, wherein the hydrophobicity of the double-stranded RNAi agent, measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent, exceeds 0.2.

52. The dsRNA agent of embodiment 51, wherein the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

53. The dsRNA agent of any of the preceding embodiments, wherein the dsRNA agent comprises at least one modified nucleotide.

54. The dsRNA agent of embodiment 53, wherein no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand are unmodified nucleotides.

55. The dsRNA agent of embodiment 53, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

56. The dsRNA agent of any one of embodiments 53-55, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxythimidine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a glycol modified nucleotide, and a 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof.

57. The dsRNA agent of any of embodiments 53-42, wherein no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand include modifications other than 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA).

58. The dsRNA agent of any of the preceding embodiments, which comprises a non-nucleotide spacer (wherein optionally the non-nucleotide spacer comprises a C3-C6 alkyl) between two of the contiguous nucleotides of the sense strand or between two of the contiguous nucleotides of the antisense strand.

59. The dsRNA agent of any of the preceding embodiments, wherein each strand is no more than 30 nucleotides in length.

60. The dsRNA agent of any of the preceding embodiments, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.

61. The dsRNA agent of any of the preceding embodiments, wherein at least one strand comprises a 3′ overhang of at least 2 nucleotides.

62. The dsRNA agent of any of the preceding embodiments, wherein the double stranded region is 15-30 nucleotide pairs in length.

63. The dsRNA agent of embodiment 62, wherein the double stranded region is 17-23 nucleotide pairs in length.

64. The dsRNA agent of embodiment 62, wherein the double stranded region is 17-25 nucleotide pairs in length.

65. The dsRNA agent of embodiment 62, wherein the double stranded region is 23-27 nucleotide pairs in length.

66. The dsRNA agent of embodiment 62, wherein the double stranded region is 19-21 nucleotide pairs in length.

67. The dsRNA agent of embodiment 62, wherein the double stranded region is 21-23 nucleotide pairs in length.

68. The dsRNA agent of any of the preceding embodiments, wherein each strand has 19-30 nucleotides.

69. The dsRNA agent of any of the preceding embodiments, wherein each strand has 19-23 nucleotides.

70. The dsRNA agent of any of the preceding embodiments, wherein each strand has 21-23 nucleotides.

71. The dsRNA agent of any of the preceding embodiments, wherein the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

72. The dsRNA agent of embodiment 71, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand.

73. The dsRNA agent of embodiment 72, wherein the strand is the antisense strand.

74. The dsRNA agent of embodiment 72, wherein the strand is the sense strand.

75. The dsRNA agent of embodiment 71, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand.

76. The dsRNA agent of embodiment 75, wherein the strand is the antisense strand.

77. The dsRNA agent of embodiment 75, wherein the strand is the sense strand.

78. The dsRNA agent of embodiment 71, wherein each of the 5′- and 3′-terminus of one strand comprises a phosphorothioate or methylphosphonate internucleotide linkage.

79. The dsRNA agent of embodiment 78, wherein the strand is the antisense strand.

80. The dsRNA agent of any of the preceding embodiments, wherein the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

81. The dsRNA agent of embodiment 78, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

82. The dsRNA agent of any one of embodiments 47-81, wherein one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand.

83. The dsRNA agent of embodiment 82, wherein the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via a linker or carrier.

84. The dsRNA agent of embodiment 83, wherein the internal positions include all positions except the terminal two positions from each end of the at least one strand.

85. The dsRNA agent of embodiment 83, wherein the internal positions include all positions except the terminal three positions from each end of the at least one strand.

86. The dsRNA agent of any one of embodiments 83-85, wherein the internal positions exclude a cleavage site region of the sense strand.

87. The dsRNA agent of embodiment 86, wherein the internal positions include all positions except positions 9-12, counting from the 5′-end of the sense strand.

88. The dsRNA agent of embodiment 86, wherein the internal positions include all positions except positions 11-13, counting from the 3′-end of the sense strand.

89. The dsRNA agent of any one of embodiments 83-85, wherein the internal positions exclude a cleavage site region of the antisense strand.

90. The dsRNA agent of embodiment 89, wherein the internal positions include all positions except positions 12-14, counting from the 5′-end of the antisense strand.

91. The dsRNA agent of any one of embodiments 83-85, wherein the internal positions include all positions except positions 11-13 on the sense strand, counting from the 3′-end, and positions 12-14 on the antisense strand, counting from the 5′-end.

92. The dsRNA agent of any one of embodiments 47-91, wherein the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′ end of each strand.

93. The dsRNA agent of embodiment 92, wherein the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5′-end of each strand.

94. The dsRNA agent of embodiment 48, wherein the positions in the double stranded region exclude a cleavage site region of the sense strand.

95. The dsRNA agent of any one of embodiments 47-80, wherein the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand.

96. The dsRNA agent of embodiment 95, wherein the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand.

97. The dsRNA agent of embodiment 95, wherein the lipophilic moiety is conjugated to position 21, position 20, or position 15 of the sense strand.

98. The dsRNA agent of embodiment 95, wherein the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand.

99. The dsRNA agent of embodiment 95, wherein the lipophilic moiety is conjugated to position 16 of the antisense strand.

100. The dsRNA agent of embodiment 95, wherein the lipophilic moiety is conjugated to position 6, counting from the 5′-end of the sense strand.

101. The dsRNA agent of any one of embodiments 47-100, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

102. The dsRNA agent of embodiment 101, wherein the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

103. The dsRNA agent of embodiment 102, wherein the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

104. The dsRNA agent of embodiment 103, wherein the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain.

105. The dsRNA agent of embodiment 103, wherein the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

106. The dsRNA agent of any one of embodiments 47-105, wherein the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region.

107. The dsRNA agent of embodiment 106, wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

108. The dsRNA agent of any one of embodiments 47-105, wherein the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.

109. The double-stranded iRNA agent of any one of embodiments 47-108, wherein the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.

110. The dsRNA agent of any one of embodiments 47-109, wherein the lipophilic moiety or targeting ligand is conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

111. The dsRNA agent of any one of embodiments 47-110, wherein the 3′ end of the sense strand is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

112. The dsRNA agent of any one of embodiments 47-111, further comprising a targeting ligand, e.g., a ligand that targets a CNS tissue or a liver tissue.

113. The dsRNA agent of embodiment 112, wherein the CNS tissue is a brain tissue or a spinal tissue.

114. The dsRNA agent of embodiment 112, wherein the targeting ligand is a GalNAc conjugate.

115. The dsRNA agent of any one of embodiments 1-114, further comprising a terminal, chiral modification occurring at the first internucleotide linkage at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.

116. The dsRNA agent of any one of embodiments 1-114, further comprising

a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

117. The dsRNA agent of any one of embodiments 1-114, further comprising

a terminal, chiral modification occurring at the first, second and third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

118. The dsRNA agent of any one of embodiments 1-114, further comprising

a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

a terminal, chiral modification occurring at the third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration,

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

119. The dsRNA agent of any one of embodiments 1-114, further comprising

a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

120. The dsRNA agent of any one of embodiments 1-119, further comprising a phosphate or phosphate mimic at the 5′-end of the antisense strand.

121. The dsRNA agent of embodiment 120, wherein the phosphate mimic is a 5′-vinyl phosphonate (VP).

122. A cell containing the dsRNA agent of any one of embodiments 1-121.

123. A human peripheral sensory neuron, e.g., (a peripheral sensory neuron in a dorsal root ganglion, or a nociceptive neuron, e.g., an A-delta fiber or a C-type fiber) comprising a reduced level of SCN9A mRNA or a level of SCN9A protein as compared to an otherwise similar untreated peripheral sensory neuron, wherein optionally the level is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

124. The human peripheral sensory neuron of embodiment 123, which was produced by a process comprising contacting a peripheral sensory neuron with the dsRNA agent of any one of embodiments 1-121.

125. A pharmaceutical composition for inhibiting expression of SCN9A, comprising the dsRNA agent of any one of embodiments 1-121.

126. A pharmaceutical composition comprising the dsRNA agent of any one of embodiments 1-121 and a lipid formulation.

127. A method of inhibiting expression of SCN9A in a cell, the method comprising:

(a) contacting the cell with the dsRNA agent of any one of embodiments 1-121, or a pharmaceutical composition of embodiment 125 or 126; and

(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of SCN9A thereby inhibiting expression of SCN9A in the cell.

128. A method of inhibiting expression of SCN9A in a cell, the method comprising:

(a) contacting the cell with the dsRNA agent of any one of embodiments 1-121, or a pharmaceutical composition of embodiment 125 or 126; and

(b) maintaining the cell produced in step (a) for a time sufficient to reduce levels of SCN9A mRNA, SCN9A protein, or both of SCN9A mRNA and protein, thereby inhibiting expression of SCN9A in the cell.

129. The method of embodiment 127 or 128, wherein the cell is within a subject.

130. The method of embodiment 129, wherein the subject is a human.

131. The method of any one of embodiments 127-130, wherein the level of SCN9A mRNA is inhibited by at least 50%.

132. The method of any one of embodiments 127-130, wherein the level of SCN9A protein is inhibited by at least 50%.

133. The method of embodiment 130-132, wherein inhibiting expression of SCN9A decreases a SCN9A protein level in a biological sample (e.g., a a cerebral spinal fluid (CSF) sample, or a CNS biopsy sample) from the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

134. The method of any one of embodiments 130-133, wherein the subject has been diagnosed with a SCN9A-associated disorder, e.g., pain, e.g., chronic pain e.g., inflammatory pain, neuropathic pain, pain hypersensitivity, pain hyposensitivity, inability to sense pain, primary erythromelalgia (PE), paroxysmal extreme pain disorder (PEPD), small fiber neuropathy (SFN), trigeminal neuralgia (TN), and pain associated with, e.g., cancer, arthritis, diabetes, traumatic injury and viral infections.

135. A method of inhibiting expression of SCN9A in an neuronal cell or tissue, the method comprising:

(a) contacting the cell or tissue with a dsRNA agent that binds SCN9A; and

(b) maintaining the cell or tissue produced in step (a) for a time sufficient to reduce levels of SCN9A mRNA, SCN9A protein, or both of SCN9A mRNA and protein, thereby inhibiting expression of SCN9A in the cell or tissue.

136. The method of embodiment 135, wherein the neuronal cell or tissue comprises a peripheral sensory neuron, e.g., a peripheral sensory neuron in a dorsal root ganglion, or a nociceptive neuron, e.g., an A-delta fiber or a C-type fiber.

137. A method of treating a subject having or diagnosed with having a SCN9A-associated disorder comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of embodiments 1-121 or a pharmaceutical composition of embodiment 125 or 126, thereby treating the disorder.

138. The method of embodiment 134 or 137, wherein the SCN9A-associated disorder is pain, e.g., chronic pain.

139. The method of embodiment 138, wherein the chronic pain is associated with one or more of the disorders in the group consisting of pain hypersensitivity, pain hyposensitivity, inability to sense pain, primary erythromelalgia (PE), paroxysmal extreme pain disorder (PEPD), small fiber neuropathy (SFN), trigeminal neuralgia (TN), or pain associated with, e.g., cancer, arthritis, diabetes, traumatic injury or viral infections.

140. The method of any one of embodiments 137-139, wherein treating comprises amelioration of at least one sign or symptom of the disorder.

141. The method of embodiment 140, wherein at least one sign or symptom of pain, e.g., chronic pain comprises a measure of one or more of pain sensitivity, pain threshold, pain level, pain disability level presence, level, or activity of SCN9A (e.g., SCN9A gene, SCN9A mRNA, or SCN9A protein).

142. The method of any one of embodiments 137-139, where treating comprises prevention of progression of the disorder.

143. The method of any one of embodiments 137-142, wherein the treating comprises one or more of (a) reducing pain; or (b) inhibiting or reducing the expression or activity of SCN9A.

144. The method of embodiment 143, wherein the treating results in at least a 30% mean reduction from baseline of SCN9A mRNA in the dorsal root ganglion.

145. The method of embodiment 144, wherein the treating results in at least a 60% mean reduction from baseline of SCN9A mRNA in dorsal root ganglion.

146. The method of embodiment 145, wherein the treating results in at least a 90% mean reduction from baseline of SCN9 mRNA in the dorsal root ganglion.

147. The method of any one of embodiments 137-146, wherein after treatment the subject experiences at least an 8-week duration of knockdown following a single dose of dsRNA as assessed by SCN9A protein in a cerebral spinal fluid (CSF) sample or a CNS biopsy sample.

148. The method of embodiment 147, wherein treating results in at least a 12-week duration of knockdown following a single dose of dsRNA as assessed by SCN9A protein in a cerebral spinal fluid (CSF) sample or a CNS biopsy sample.

149. The method of embodiment 148, wherein treating results in at least a 16-week duration of knockdown following a single dose of dsRNA as assessed by SCN9A protein in a cerebral spinal fluid (CSF) sample or a CNS biopsy sample.

150. The method of any of embodiments 129-149, wherein the subject is human.

151. The method of any one of embodiments 130-150, wherein the dsRNA agent is administered at a dose of about 0.01 mg/kg to about 50 mg/kg.

152. The method of any one of embodiments 130-151, wherein the dsRNA agent is administered to the subject intracranially or intrathecally,

153. The method of any one of embodiments 130-151, wherein the dsRNA agent is administered to the subject intrathecally, intraventricularly, or intracerebrally.

154. The method of any one of embodiments 130-153, further comprising measuring level of SCN9A (e.g., SCN9A gene, SCN9A mRNA, or SCN9A protein) in the subject.

155. The method of embodiment 154, where measuring the level of SCN9A in the subject comprises measuring the level of SCN9A gene, SCN9A protein or SCN9A mRNA in a biological sample from the subject (e.g., a cerebral spinal fluid (CSF) sample or a CNS biopsy sample).

156. The method of any one of embodiments 130-155, further comprising performing a blood test, an imaging test, a CNS biopsy sample, or an aqueous cerebral spinal fluid biopsy.

157. The method of any one of embodiments 154-156, wherein measuring level of SCN9A (e.g., SCN9A gene, SCN9A mRNA, or SCN9A protein) in the subject is performed prior to treatment with the dsRNA agent or the pharmaceutical composition.

158. The method of embodiment 157, wherein, upon determination that a subject has a level of SCN9A (e.g., SCN9A gene, SCN9A mRNA, or SCN9A protein) that is greater than a reference level, the dsRNA agent or the pharmaceutical composition is administered to the subject.

159. The method of any one of embodiments 155-158, wherein measuring level of SCN9A (e.g., SCN9A gene, SCN9A mRNA, or SCN9A protein) in the subject is performed after treatment with the dsRNA agent or the pharmaceutical composition.

160. The method of any one of embodiments 137-159, further comprising administering to the subject an additional agent and/or therapy suitable for treatment or prevention of an SCN9A-associated disorder.

161. The method of embodiment 160, wherein the additional agent and/or therapy comprises one or more of a non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen, opioids, or corticosteroids, acupuncture, therapeutic massage, dorsal root ganglion stimulation, spinal cord stimulation, or topical pain relievers.

EXAMPLES Example 1. SCN9A siRNA

Nucleic acid sequences provided herein are represented using standard nomenclature. See the abbreviations of Table 1.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acid sequence representation It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds; and it is understood that when the nucleotide contains a 2′-fluoro modification, then the fluoro replaces the hydroxy at that position in the parent nucleotide (i.e., it is a 2′-deoxy-2′-fluoronucleotide). . . Abbreviation Nucleotide(s) A Adenosine-3′-phosphate Ab beta-L-adenosine-3′-phosphate Abs beta-L-adenosine-3′-phosphorothioate Af 2′-fluoroadenosine-3′-phosphate Afs 2′-fluoroadenosine-3′-phosphorothioate (Ahd) 2′-O-hexadecyl-adenosine-3′-phosphate (Ahds) 2′-O-hexadecyl-adenosine-3′-phosphorothioate As adenosine-3′-phosphorothioate (A2p) adenosine 2′-phosphate C cytidine-3′-phosphate Cb beta-L-cytidine-3′-phosphate Cbs beta-L-cytidine-3′-phosphorothioate Cf 2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioate (Chd) 2′-O-hexadecyl-cytidine-3′-phosphate (Chds) 2′-O-hexadecyl-cytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate (C2p) cytosine 2′-phosphate G guanosine-3′-phosphate Gb beta-L-guanosine-3′-phosphate Gbs beta-L-guanosine-3′-phosphorothioate Gf 2′-fluoroguanosine-3′-phosphate Gfs 2′-fluoroguanosine-3′-phosphorothioate (Ghd) 2′-O-hexadecyl-guanosine-3′-phosphate (Ghds) 2′-O-hexadecyl-guanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioate (G2p) guanosine 2′-phosphate T 5′-methyluridine-3′-phosphate Tb beta-L-thymidine-3′-phosphate Tbs beta-L-thymidine-3′-phosphorothioate Tf 2′-fluoro-5-methyluridine-3′-phosphate Tfs 2′-fluoro-5-methyluridine-3′-phosphorothioate Tgn thymidine-glycol nucleic acid (GNA) S-Isomer Agn adenosine-glycol nucleic acid (GNA) S-Isomer Cgn cytidine-glycol nucleic acid (GNA) S-Isomer Ggn guanosine-glycol nucleic acid (GNA) S-Isomer Ts 5-methyluridine-3′-phosphorothioate (T2p) thymidine 2′-phosphate U Uridine-3′-phosphate Ub beta-L-uridine-3′-phosphate Ubs beta-L-uridine-3′-phosphorothioate Uf 2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine -3′-phosphorothioate (Uhd) 2′-O-hexadecyl-uridine-3′-phosphate (Uhds) 2′-O-hexadecyl-uridine-3′-phosphorothioate Us uridine -3′-phosphorothioate (U2p) uracil 2′-phosphate N any nucleotide (G, A, C, T or U) VP Vinyl phosphonate a 2′-O-methyladenosine-3′-phosphate as 2′-O-methyladenosine-3′-phosphorothioate c 2′-O-methylcytidine-3′-phosphate cs 2′-O-methylcytidine-3′-phosphorothioate g 2′-O-methylguanosine-3′-phosphate gs 2′-O-methylguanosine-3′-phosphorothioate t 2′-O-methyl-5-methyluridine-3′-phosphate ts 2′-O-methyl-5-methyluridine-3′-phosphorothioate u 2′-O-methyluridine-3′-phosphate us 2′-O-methyluridine-3′-phosphorothioate dA 2′-deoxyadenosine-3′-phosphate dAs 2′-deoxyadenosine-3′-phosphorothioate dC 2′-deoxycytidine-3′-phosphate dCs 2′-deoxycytidine-3′-phosphorothioate dG 2′-deoxyguanosine-3′-phosphate dGs 2′-deoxyguanosine-3′-phosphorothioate dT 2′-deoxythymidine dTs 2′-deoxythymidine-3′-phosphorothioate dU 2′-deoxyuridine s phosphorothioate linkage L96¹ N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol Hyp-(GalNAc-alkyl)3 (Aeo) 2′-O-methoxyethyladenosine-3′-phosphate (Aeos) 2′-O-methoxyethyladenosine-3′-phosphorothioate (Geo) 2′-O-methoxyethylguanosine-3′-phosphate (Geos) 2′-O-methoxyethylguanosine-3′-phosphorothioate (Teo) 2′-O-methoxyethyl-5-methyluridine-3′-phosphate (Teos) 2′-O-methoxyethyl-5-methyluridine-3′-phosphorothioate (m5Ceo) 2′-O-methoxyethyl-5-methylcytidine-3′-phosphate (m5Ceos) 2′-O-methoxyethyl-5-methylcytidine-3′-phosphorothioate ¹The chemical structure of L96 is as follows:

Experimental Methods Bioinformatics

Transcripts

A set of siRNAs targeting the human SCN9A, “sodium channel, voltage gated, type IX alpha subunit” (human NCBI refseqID NM_002977.3; NCBI GeneID: 6335 or human: NCBI refseqID NM_001365536.1; NCBI GeneID: 6335) were generated. The human NM_002977.3 REFSEQ mRNA, has a length of 9771 bases. The human NM_001365536.1 REFSEQ mRNA, has a length of 9752 bases. Pairs of oligos were generated using bioinformatic methods and ranked, and exemplary pairs of oligos are shown in Table 2A, Table 2B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 13A, Table 13B, Table 14A, Table 14B, Table 15A, Table 15B, and Table 16. Modified sequences are presented in Table 2A, Table 4A, Table 5A, Table 6A, Table 13A, Table 14A, Table 15A, and Table 16. Unmodified sequences are presented in Table 2B, Table 4B, Table 5B, Table 6B, Table 13B, Table 14B, and Table 15B. The target mRNA source for each exemplary set of duplexes is in Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, and 16 are denoted in the tables. The number following the decimal point in a duplex name as indicated in the tables merely refers to a batch production number.

TABLE 2A Exemplary Human SCN9A siRNA Modified Single Strands and Duplex Sequences Column 1 indicates duplex name. Column 2 indicates the name of the sense sequence. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the modified sequence of a sense strand suitable for use in a duplex described herein. Column 5 indicates the antisense sequence name. Column 6 indicates the sequence ID for the sequence of column 7. Column 7 provides the sequence of a modified antisense strand suitable for use in a duplex described herein, e.g., a duplex comprising the sense sequence in the same row of the table. Column 8 indicates the position in the target mRNA (NM_002977.3) that is complementary to the antisense strand of Column 7. Column9  indicated the sequence ID for the sequence of column 8. Sense Seq ID Antisense Seq ID mRNA target Duplex sequence NO: Sense sequence sequence NO: Antisense sequence sequence in Seq ID NO: Name name (sense) (5′-3′) name (antisense) (5′-3′) NM_002977.3 (mRNA target) AD- A- 3 UCACAAAACAGU A-1683739.1 4 GCAAGAGACUGUUU UCACAAAACAGUCUC 3039 887232 1683738. CUCUUGCdTdT UGUGAdTdT UUGC 1 AD- A- 5 GGAAAACAAUCU A-1683741.1 6 AAACGGAAGAUUGU GGAAAACAAUCUUCC 3040 887233 1683740. UCCGUUUdTdT UUUCCdTdT GUUU 1 AD- A- 7 GAAAACAAUCUU A-1683743.1 8 GAAACGGAAGAUUG GAAAACAAUCUUCCG 3041 887234 1683742. CCGUUUCdTdT UUUUCdTdT UUUC 1 AD- A- 9 AAAACAAUCUUC A-1683745.1 10 UGAAACGGAAGAUU AAAACAAUCUUCCGU 3042 887235 1683744. CGUUUCAdTdT GUUUUdTdT UUCA 1 AD- A- 11 AAACAAUCUUCC A-1683747.1 12 UUGAAACGGAAGAU AAACAAUCUUCCGUU 3043 887236 1683746. GUUUCAAdTdT UGUUUdTdT UCAA 1 AD- A- 13 AACAAUCUUCCG A-1683749.1 14 AUUGAAACGGAAGA AACAAUCUUCCGUUU 3044 887237 1683748. UUUCAAUdTdT UUGUUdTdT CAAU 1 AD- A- 15 CAAUCUUCCGUU A-1683751.1 16 GCAUUGAAACGGAA CAAUCUUCCGUUUCA 3045 887238 1683750. UCAAUGCdTdT GAUUGdTdT AUGC 1 AD- A- 17 CCUGCUUUAUAU A-1683753.1 18 AAAGCAUAUAUAAA CCUGCUUUAUAUAUG 3046 887239 1683752. AUGCUUUdTdT GCAGGdTdT CUUU 1 AD- A- 19 CUGCUUUAUAUA A-1683755.1 20 GAAAGCAUAUAUAA CUGCUUUAUAUAUGC 3047 887240 1683754. UGCUUUCdTdT AGCAGdTdT UUUC 1 AD- A- 21 UAUGCUUUCUCC A-1683757.1 22 ACUGAAAGGAGAAA UAUGCUUUCUCCUUU 3048 887241 1683756. UUUCAGUdTdT GCAUAdTdT CAGU 1 AD- A- 23 AUGCUUUCUCCU A-1683759.1 24 GACUGAAAGGAGAA AUGCUUUCUCCUUUC 3049 887242 1683758. UUCAGUCdTdT AGCAUdTdT AGUC 1 AD- A- 25 UGCUUUCUCCUU A-1683761.1 26 GGACUGAAAGGAGA UGCUUUCUCCUUUCA 3050 887243 1683760. UCAGUCCdTdT AAGCAdTdT GUCC 1 AD- A- 27 cuuucuccuuuc A-1683763.1 28 GAGGACUGAAAGGA CUUUCUCCUUUCAGU 3051 887244 1683762. AGUCCUCdTdT GAAAGdTdT CCUC 1 AD- A- 29 UCUCCUUUCAGU A-1683765.1 30 UUAGAGGACUGAAA UCUCCUUUCAGUCCU 3052 887245 1683764. CCUCUAAdTdT GGAGAdTdT CUAA 1 AD- A- 31 CUCCUUUCAGUC A-1683767.1 32 CUUAGAGGACUGAA CUCCUUUCAGUCCUC 3053 887246 1683766. CUCUAAGdTdT AGGAGdTdT UAAG 1 AD- A- 33 UCCUUUCAGUCC A-1683769.1 34 UCUUAGAGGACUGA UCCUUUCAGUCCUCU 3054 887247 1683768. UCUAAGAdTdT AAGGAdTdT AAGA 1 AD- A- 35 CCUUUCAGUCCU A-1683771.1 36 UUCUUAGAGGACUG CCUUUCAGUCCUCUA 3055 887248 1683770. CUAAGAAdTdT AAAGGdTdT AGAA 1 AD- A- 37 CUUUCAGUCCUC A-1683773.1 38 CUUCUUAGAGGACU CUUUCAGUCCUCUAA 3056 887249 1683772. UAAGAAGdTdT GAAAGdTdT GAAG 1 AD- A- 39 AGUCCUCUAAGA A-1683775.1 40 AUAUUCUUCUUAGA AGUCCUCUAAGAAGA 3057 887250 1683774. AGAAUAUdTdT GGACUdTdT AUAU 1 AD- A- 41 UCCUCUAAGAAG A-1683777.1 42 AGAUAUUCUUCUUA UCCUCUAAGAAGAAU 3058 887251 1683776. AAUAUCUdTdT GAGGAdTdT AUCU 1 AD- A- 43 CCUCUAAGAAGA A-1683779.1 44 UAGAUAUUCUUCUU CCUCUAAGAAGAAUA 3059 887252 1683778. AUAUCUAdTdT AGAGGdTdT UCUA 1 AD- A- 45 CUCUAAGAAGAA A-1683781.1 46 AUAGAUAUUCUUCU CUCUAAGAAGAAUAU 3060 887253 1683780. UAUCUAUdTdT UAGAGdTdT CUAU 1 AD- A- 47 AUUUUAGUACAC A-1683783.1 48 AUAAGGAGUGUACU AUUUUAGUACACUCC 3061 887254 1683782. UCCUUAUdTdT AAAAUdTdT UUAU 1 AD- A- 49 UAGUACACUCCU A-1683785.1 50 CUGAAUAAGGAGUG UAGUACACUCCUUAU 3062 887255 1683784. UAUUCAGdTdT UACUAdTdT UCAG 1 AD- A- 51 AGUACACUCCUU A-1683787.1 52 GCUGAAUAAGGAGU AGUACACUCCUUAUU 3063 887256 1683786. AUUCAGCdTdT GUACUdTdT CAGC 1 AD- A- 53 CCUUAUUCAGCA A-1683789.1 54 AUGAGCAUGCUGAA CCUUAUUCAGCAUGC 3064 887257 1683788. UGCUCAUdTdT UAAGGdTdT UCAU 1 AD- A- 55 UCAUCAUGUGCA A-1683791.1 56 AGAAUAGUGCACAU UCAUCAUGUGCACUA 3065 887258 1683790. CUAUUCUdTdT GAUGAdTdT UUCU 1 AD- A- 57 CAUCAUGUGCAC A-1683793.1 58 CAGAAUAGUGCACAU CAUCAUGUGCACUAU 3066 887259 1683792. UAUUCUGdTdT GAUGdTdT UCUG 1 AD- A- 59 UGUCGAGUACAC A-1683795.1 60 AGUAAAAGUGUACU UGUCGAGUACACUUU 3067 887260 1683794. UUUUACUdTdT CGACAdTdT UACU 1 AD- A- 61 GUCGAGUACACU A-1683797.1 62 CAGUAAAAGUGUAC GUCGAGUACACUUUU 3068 887261 1683796. UUUACUGdTdT UCGACdTdT ACUG 1 AD- A- 63 CUUCUGUGUAG A-1683799.1 64 GAAUUCUCCUACACA CUUCUGUGUAGGAGA 3069 887262 1683798. GAGAAUUCdTdT GAAGdTdT AUUC 1 AD- A- 65 UAGGAGAAUUCA A-1683801.1 66 AGAAAAGUGAAUUC UAGGAGAAUUCACUU 3070 887263 1683800. CUUUUCUdTdT UCCUAdTdT UUCU 1 AD- A- 67 AGGAGAAUUCAC A-1683803.1 68 AAGAAAAGUGAAUU AGGAGAAUUCACUUU 3071 887264 1683802. UUUUCUUdTdT CUCCUdTdT UCUU 1 AD- A- 69 GGAGAAUUCACU A-1683805.1 70 GAAGAAAAGUGAAU GGAGAAUUCACUUUU 3072 887265 1683804. UUUCUUCdTdT UCUCCdTdT CUUC 1 AD- A- 71 GGCAAUGUUUCA A-1683807.1 72 GAAGAGCUGAAACA GGCAAUGUUUCAGCU 3073 887266 1683806. GCUCUUCdTdT UUGCCdTdT CUUC 1 AD- A- 73 AAUGUUUCAGCU A-1683809.1 74 UUCGAAGAGCUGAA AAUGUUUCAGCUCUU 3074 887267 1683808. CUUCGAAdTdT ACAUUdTdT CGAA 1 AD- A- 75 GUUUCAGCUCUU A-1683811.1 76 AAGUUCGAAGAGCU GUUUCAGCUCUUCGA 3075 887268 1683810. CGAACUUdTdT GAAACdTdT ACUU 1 AD- A- 77 UCAGCUCUUCGA A-1683813.1 78 UGAAAGUUCGAAGA UCAGCUCUUCGAACU 3076 887269 1683812. ACUUUCAdTdT GCUGAdTdT UUCA 1 AD- A- 79 AGCUCUUCGAAC A-1683815.1 80 UCUGAAAGUUCGAA AGCUCUUCGAACUUU 3077 887270 1683814. UUUCAGAdTdT GAGCUdTdT CAGA 1 AD- A- 81 CUCUUCGAACUU A-1683817.1 82 ACUCUGAAAGUUCG CUCUUCGAACUUUCA 3078 887271 1683816. UCAGAGUdTdT AAGAGdTdT GAGU 1 AD- A- 83 CUUCGAACUUUC A-1683819.1 84 AUACUCUGAAAGUU CUUCGAACUUUCAGA 3079 887272 1683818. AGAGUAUdTdT CGAAGdTdT GUAU 1 AD- A- 85 UCCUGACUGUGU A-1683821.1 86 AGACAGAACACAGUC UCCUGACUGUGUUCU 3080 887273 1683820. UCUGUCUdTdT AGGAdTdT GUCU 1 AD- A- 87 CUGACUGUGUUC A-1683823.1 88 UCAGACAGAACACAG CUGACUGUGUUCUGU 3081 887274 1683822. UGUCUGAdTdT UCAGdTdT CUGA 1 AD- A- 89 UGACUGUGUUC A-1683825.1 90 CUCAGACAGAACACA UGACUGUGUUCUGUC 3082 887275 1683824. UGUCUGAGdTdT GUCAdTdT UGAG 1 AD- A- 91 GACUGUGUUCU A-1683827.1 92 ACUCAGACAGAACAC GACUGUGUUCUGUCU 3083 887276 1683826. GUCUGAGUdTdT AGUCdTdT GAGU 1 AD- A- 93 ACUGUGUUCUG A-1683829.1 94 CACUCAGACAGAACA ACUGUGUUCUGUCUG 3084 887277 1683828. UCUGAGUGdTdT CAGUdTdT AGUG 1 AD- A- 95 CUGUGUUCUGUC A-1683831.1 96 ACACUCAGACAGAAC CUGUGUUCUGUCUGA 3085 887278 1683830. UGAGUGUdTdT ACAGdTdT GUGU 1 AD- A- 97 UGUGUUCUGUC A-1683833.1 98 CACACUCAGACAGAA UGUGUUCUGUCUGA 3086 887279 1683832. UGAGUGUGdTdT CACAdTdT GUGUG 1 AD- A- 99 UGUUCUGUCUG A-1683835.1 100 AACACACUCAGACAG UGUUCUGUCUGAGU 3087 887280 1683834. AGUGUGUUdTdT AACAdTdT GUGUU 1 AD- A- 101 GUUCUGUCUGA A-1683837.1 102 AAACACACUCAGACA GUUCUGUCUGAGUG 3088 887281 1683836. GUGUGUUUdTdT GAACdTdT UGUUU 1 AD- A- 103 UUCUGUCUGAG A-1683839.1 104 CAAACACACUCAGAC UUCUGUCUGAGUGU 3089 887282 1683838. UGUGUUUGdTdT AGAAdTdT GUUUG 1 AD- A- 105 UCUGUCUGAGU A-1683841.1 106 GCAAACACACUCAGA UCUGUCUGAGUGUG 3090 887283 1683840. GUGUUUGCdTdT CAGAdTdT UUUGC 1 AD- A- 107 UGCUCUCCUUUG A-1683843.1 108 GAAACCACAAAGGAG UGCUCUCCUUUGUGG 3091 887284 1683842. UGGUUUCdTdT AGCAdTdT UUUC 1 AD- A- 109 CUCUCCUUUGUG A-1683845.1 110 CUGAAACCACAAAGG CUCUCCUUUGUGGUU 3092 887285 1683844. GUUUCAGdTdT AGAGdTdT UCAG 1 AD- A- 111 UCUCCUUUGUGG A-1683847.1 112 GCUGAAACCACAAAG UCUCCUUUGUGGUU 3093 887286 1683846. UUUCAGCdTdT GAGAdTdT UCAGC 1 AD- A- 113 CUCCUUUGUGGU A-1683849.1 114 UGCUGAAACCACAAA CUCCUUUGUGGUUUC 3094 887287 1683848. UUCAGCAdTdT GGAGdTdT AGCA 1 AD- A- 115 CGAGCUUUGACA A-1683851.1 116 CUGAAAGUGUCAAA CGAGCUUUGACACUU 3095 887288 1683850. CUUUCAGdTdT GCUCGdTdT UCAG 1 AD- A- 117 ACAUGAUCUUCU A-1683853.1 118 ACGACAAAGAAGAUC ACAUGAUCUUCUUUG 3096 887289 1683852. UUGUCGUdTdT AUGUdTdT UCGU 1 AD- A- 119 CAUGAUCUUCUU A-1683855.1 120 UACGACAAAGAAGAU CAUGAUCUUCUUUGU 3097 887290 1683854. UGUCGUAdTdT CAUGdTdT CGUA 1 AD- A- 121 GAUCUUCUUUG A-1683857.1 122 CACUACGACAAAGAA GAUCUUCUUUGUCGU 3098 887291 1683856. UCGUAGUGdTdT GAUCdTdT AGUG 1 AD- A- 123 UCUUCUUUGUCG A-1683859.1 124 AUCACUACGACAAAG UCUUCUUUGUCGUAG 3099 887292 1683858. UAGUGAUdTdT AAGAdTdT UGAU 1 AD- A- 125 CUUCUUUGUCGU A-1683861.1 126 AAUCACUACGACAAA CUUCUUUGUCGUAGU 3100 887293 1683860. AGUGAUUdTdT GAAGdTdT GAUU 1 AD- A- 127 UUGUCGUAGUG A-1683863.1 128 AGGAAAAUCACUACG UUGUCGUAGUGAUU 3101 887294 1683862. AUUUUCCUdTdT ACAAdTdT UUCCU 1 AD- A- 129 GCUCCUUUUAUC A-1683865.1 130 UUUAUUAGAUAAAA GCUCCUUUUAUCUAA 3102 887295 1683864. UAAUAAAdTdT GGAGCdTdT UAAA 1 AD- A- 131 CUCCUUUUAUCU A-1683867.1 132 GUUUAUUAGAUAAA CUCCUUUUAUCUAAU 3103 887296 1683866. AAUAAACdTdT AGGAGdTdT AAAC 1 AD- A- 133 CCUCUCAGAGAG A-1683869.1 134 AGAAGAACUCUCUGA CCUCUCAGAGAGUUC 3104 887297 1683868. UUCUUCUdTdT GAGGdTdT UUCU 1 AD- A- 135 CUCUCAGAGAGU A-1683871.1 136 CAGAAGAACUCUCUG CUCUCAGAGAGUUCU 3105 887298 1683870. UCUUCUGdTdT AGAGdTdT UCUG 1 AD- A- 137 UCUCAGAGAGUU A-1683873.1 138 UCAGAAGAACUCUCU UCUCAGAGAGUUCUU 3106 887299 1683872. CUUCUGAdTdT GAGAdTdT CUGA 1 AD- A- 139 CUCAGAGAGUUC A-1683875.1 140 UUCAGAAGAACUCUC CUCAGAGAGUUCUUC 3107 887300 1683874. UUCUGAAdTdT UGAGdTdT UGAA 1 AD- A- 141 UCAGAGAGUUCU A-1683877.1 142 UUUCAGAAGAACUC UCAGAGAGUUCUUCU 3108 887301 1683876. UCUGAAAdTdT UCUGAdTdT GAAA 1 AD- A- 143 CAGAGAGUUCUU A-1683879.1 144 GUUUCAGAAGAACU CAGAGAGUUCUUCUG 3109 887302 1683878. CUGAAACdTdT CUCUGdTdT AAAC 1 AD- A- 145 GAGAGUUCUUCU A-1683881.1 146 AUGUUUCAGAAGAA GAGAGUUCUUCUGAA 3110 887303 1683880. GAAACAUdTdT CUCUCdTdT ACAU 1 AD- A- 147 AGAGUUCUUCUG A-1683883.1 148 GAUGUUUCAGAAGA AGAGUUCUUCUGAAA 3111 887304 1683882. AAACAUCdTdT ACUCUdTdT CAUC 1 AD- A- 149 GAGUUCUUCUGA A-1683885.1 150 GGAUGUUUCAGAAG GAGUUCUUCUGAAAC 3112 887305 1683884. AACAUCCdTdT AACUCdTdT AUCC 1 AD- A- 151 AGUUCUUCUGAA A-1683887.1 152 UGGAUGUUUCAGAA AGUUCUUCUGAAACA 3113 887306 1683886. ACAUCCAdTdT GAACUdTdT UCCA 1 AD- A- 153 GUUCUUCUGAAA A-1683889.1 154 UUGGAUGUUUCAGA GUUCUUCUGAAACAU 3114 887307 1683888. CAUCCAAdTdT AGAACdTdT CCAA 1 AD- A- 155 UCUUCUGAAACA A-1683891.1 156 GUUUGGAUGUUUCA UCUUCUGAAACAUCC 3115 887308 1683890. UCCAAACdTdT GAAGAdTdT AAAC 1 AD- A- 157 CUUCUGAAACAU A-1683893.1 158 AGUUUGGAUGUUUC CUUCUGAAACAUCCA 3116 887309 1683892. CCAAACUdTdT AGAAGdTdT AACU 1 AD- A- 159 UCUGAAACAUCC A-1683895.1 160 UCAGUUUGGAUGUU UCUGAAACAUCCAAA 3117 887310 1683894. AAACUGAdTdT UCAGAdTdT CUGA 1 AD- A- 161 UCCAAACUGAGC A-1683897.1 162 UUUUAGAGCUCAGU UCCAAACUGAGCUCU 3118 887311 1683896. UCUAAAAdTdT UUGGAdTdT AAAA 1 AD- A- 163 AGGCGUUGUAG A-1683899.1 164 GAUAGGAACUACAAC AGGCGUUGUAGUUCC 3119 887312 1683898. UUCCUAUCdTdT GCCUdTdT UAUC 1 AD- A- 165 GCGUUGUAGUU A-1683901.1 166 GAGAUAGGAACUAC GCGUUGUAGUUCCUA 3120 887313 1683900. CCUAUCUCdTdT AACGCdTdT UCUC 1 AD- A- 167 CGUUGUAGUUCC A-1683903.1 168 GGAGAUAGGAACUA CGUUGUAGUUCCUAU 3121 887314 1683902. UAUCUCCdTdT CAACGdTdT CUCC 1 AD- A- 169 GUUGUAGUUCC A-1683905.1 170 AGGAGAUAGGAACU GUUGUAGUUCCUAUC 3122 887315 1683904. UAUCUCCUdTdT ACAACdTdT uccu 1 AD- A- 171 UUGUAGUUCCUA A-1683907.1 172 AAGGAGAUAGGAAC UUGUAGUUCCUAUCU 3123 887316 1683906. UCUCCUUdTdT UACAAdTdT CCUU 1 AD- A- 173 UGUAGUUCCUAU A-1683909.1 174 AAAGGAGAUAGGAA UGUAGUUCCUAUCUC 3124 887317 1683908. CUCCUUUdTdT CUACAdTdT CUUU 1 AD- A- 175 GUAGUUCCUAUC A-1683911.1 176 GAAAGGAGAUAGGA GUAGUUCCUAUCUCC 3125 887318 1683910. UCCUUUCdTdT ACUACdTdT UUUC 1 AD- A- 177 UAGUUCCUAUCU A-1683913.1 178 UGAAAGGAGAUAGG UAGUUCCUAUCUCCU 3126 887319 1683912. CCUUUCAdTdT AACUAdTdT UUCA 1 AD- A- 179 AGUUCCUAUCUC A-1683915.1 180 CUGAAAGGAGAUAG AGUUCCUAUCUCCUU 3127 887320 1683914. CUUUCAGdTdT GAACUdTdT UCAG 1 AD- A- 181 GUUCCUAUCUCC A-1683917.1 182 UCUGAAAGGAGAUA GUUCCUAUCUCCUUU 3128 887321 1683916. UUUCAGAdTdT GGAACdTdT CAGA 1 AD- A- 183 UUCCUAUCUCCU A-1683919.1 184 CUCUGAAAGGAGAU UUCCUAUCUCCUUUC 3129 887322 1683918. UUCAGAGdTdT AGGAAdTdT AGAG 1 AD- A- 185 UCCUAUCUCCUU A-1683921.1 186 CCUCUGAAAGGAGA UCCUAUCUCCUUUCA 3130 887323 1683920. UCAGAGGdTdT UAGGAdTdT GAGG 1 AD- A- 187 UCUCCUUUCAGA A-1683923.1 188 CAUAUCCUCUGAAAG UCUCCUUUCAGAGGA 3131 887324 1683922. GGAUAUGdTdT GAGAdTdT UAUG 1 AD- A- 189 GCAUAUUAACAA A-1683925.1 190 ACAGUGUUUGUUAA GCAUAUUAACAAACA 3132 887325 1683924. ACACUGUdTdT UAUGCdTdT CUGU 1 AD- A- 191 CUUGAUCUGGAA A-1683927.1 192 AGAGCAAUUCCAGAU CUUGAUCUGGAAUUG 3133 887326 1683926. UUGCUCUdTdT CAAGdTdT CUCU 1 AD- A- 193 CUCUCCAUAUUG A-1683929.1 194 UUUUAUCCAAUAUG CUCUCCAUAUUGGAU 3134 887327 1683928. GAUAAAAdTdT GAGAGdTdT AAAA 1 AD- A- 195 UCUCCAUAUUGG A-1683931.1 196 AUUUUAUCCAAUAU UCUCCAUAUUGGAUA 3135 887328 1683930. AUAAAAUdTdT GGAGAdTdT AAAU 1 AD- A- 197 CUCCAUAUUGGA A-1683933.1 198 AAUUUUAUCCAAUA CUCCAUAUUGGAUAA 3136 887329 1683932. UAAAAUUdTdT UGGAGdTdT AAUU 1 AD- A- 199 GAUCUUGCAAUU A-1683935.1 200 AAAUGGUAAUUGCA GAUCUUGCAAUUACC 3137 887330 1683934. ACCAUUUdTdT AGAUCdTdT AUUU 1 AD- A- 201 UUGGUCUUUAC A-1683937.1 202 AGAUUCCAGUAAAG UUGGUCUUUACUGGA 3138 887331 1683936. UGGAAUCUdTdT ACCAAdTdT AUCU 1 AD- A- 203 GGUCUUUACUG A-1683939.1 204 AAAGAUUCCAGUAAA GGUCUUUACUGGAAU 3139 887332 1683938. GAAUCUUUdTdT GACCdTdT CUUU 1 AD- A- 205 GUCUUUACUGGA A-1683941.1 206 CAAAGAUUCCAGUAA GUCUUUACUGGAAUC 3140 887333 1683940. AUCUUUGdTdT AGACdTdT UUUG 1 AD- A- 207 GCCUUAUUGUGA A-1683943.1 208 CUUAAAGUCACAAUA GCCUUAUUGUGACUU 3141 887334 1683942. CUUUAAGdTdT AGGCdTdT UAAG 1 AD- A- 209 GCUCUUUCUAGC A-1683945.1 210 CACAUCUGCUAGAAA GCUCUUUCUAGCAGA 3142 887335 1683944. AGAUGUGdTdT GAGCdTdT UGUG 1 AD- A- 211 CUCUUUCUAGCA A-1683947.1 212 CCACAUCUGCUAGAA CUCUUUCUAGCAGAU 3143 887336 1683946. GAUGUGGdTdT AGAGdTdT GUGG 1 AD- A- 213 GUCAGUUCUGCG A-1683949.1 214 GAAUGAUCGCAGAAC GUCAGUUCUGCGAUC 3144 887337 1683948. AUCAUUCdTdT UGACdTdT AUUC 1 AD- A- 215 UCAGUUCUGCGA A-1683951.1 216 UGAAUGAUCGCAGA UCAGUUCUGCGAUCA 3145 887338 1683950. UCAUUCAdTdT ACUGAdTdT UUCA 1 AD- A- 217 AGUCUUCAAGUU A-1683953.1 218 UUUUGCCAACUUGA AGUCUUCAAGUUGGC 3146 887339 1683952. GGCAAAAdTdT AGACUdTdT AAAA 1 AD- A- 219 UCUUCAAGUUGG A-1683955.1 220 GAUUUUGCCAACUU UCUUCAAGUUGGCAA 3147 887340 1683954. CAAAAUCdTdT GAAGAdTdT AAUC 1 AD- A- 221 CUUCAAGUUGGC A-1683957.1 222 GGAUUUUGCCAACU CUUCAAGUUGGCAAA 3148 887341 1683956. AAAAUCCdTdT UGAAGdTdT AUCC 1 AD- A- 223 CCAUCAUCGUCU A-1683959.1 224 AAAAUGAAGACGAU CCAUCAUCGUCUUCA 3149 887342 1683958. UCAUUUUdTdT GAUGGdTdT UUUU 1 AD- A- 225 CAUCAUCGUCUU A-1683961.1 226 AAAAAUGAAGACGAU CAUCAUCGUCUUCAU 3150 887343 1683960. CAUUUUUdTdT GAUGdTdT UUUU 1 AD- A- 227 GCACAUGAACGA A-1683963.1 228 GAAGAAGUCGUUCA GCACAUGAACGACUU 3151 887344 1683962. CUUCUUCdTdT UGUGCdTdT CUUC 1 AD- A- 229 CACAUGAACGAC A-1683965.1 230 GGAAGAAGUCGUUC CACAUGAACGACUUC 3152 887345 1683964. UUCUUCCdTdT AUGUGdTdT UUCC 1 AD- A- 231 ACAUGAACGACU A-1683967.1 232 UGGAAGAAGUCGUU ACAUGAACGACUUCU 3153 887346 1683966. UCUUCCAdTdT CAUGUdTdT UCCA 1 AD- A- 233 CAUGAACGACUU A-1683969.1 234 GUGGAAGAAGUCGU CAUGAACGACUUCUU 3154 887347 1683968. CUUCCACdTdT UCAUGdTdT CCAC 1 AD- A- 235 UGAACGACUUCU A-1683971.1 236 GAGUGGAAGAAGUC UGAACGACUUCUUCC 3155 887348 1683970. UCCACUCdTdT GUUCAdTdT ACUC 1 AD- A- 237 CGACUUCUUCCA A-1683973.1 238 GAAGGAGUGGAAGA CGACUUCUUCCACUC 3156 887349 1683972. CUCCUUCdTdT AGUCGdTdT CUUC 1 AD- A- 239 UCCACUCCUUCC A-1683975.1 240 ACAAUCAGGAAGGAG UCCACUCCUUCCUGA 3157 887350 1683974. UGAUUGUdTdT UGGAdTdT UUGU 1 AD- A- 241 ACUCCUUCCUGA A-1683977.1 242 AACACAAUCAGGAAG ACUCCUUCCUGAUUG 3158 887351 1683976. UUGUGUUdTdT GAGUdTdT UGUU 1 AD- A- 243 CUCCUUCCUGAU A-1683979.1 244 GAACACAAUCAGGAA CUCCUUCCUGAUUGU 3159 887352 1683978. UGUGUUCdTdT GGAGdTdT GUUC 1 AD- A- 245 UCCUUCCUGAUU A-1683981.1 246 GGAACACAAUCAGGA UCCUUCCUGAUUGUG 3160 887353 1683980. GUGUUCCdTdT AGGAdTdT UUCC 1 AD- A- 247 CUAUGUGCCUUA A-1683983.1 248 UAAACAAUAAGGCAC CUAUGUGCCUUAUUG 3161 887354 1683982. UUGUUUAdTdT AUAGdTdT UUUA 1 AD- A- 249 UGGUCCUAAACC A-1683985.1 250 AGAAAUAGGUUUAG UGGUCCUAAACCUAU 3162 887355 1683984. UAUUUCUdTdT GACCAdTdT UUCU 1 AD- A- 251 GGUCCUAAACCU A-1683987.1 252 CAGAAAUAGGUUUA GGUCCUAAACCUAUU 3163 887356 1683986. AUUUCUGdTdT GGACCdTdT UCUG 1 AD- A- 253 GUCCUAAACCUA A-1683989.1 254 CCAGAAAUAGGUUU GUCCUAAACCUAUUU 3164 887357 1683988. UUUCUGGdTdT AGGACdTdT CUGG 1 AD- A- 255 CCUUACGUGAAU A-1683991.1 256 AGAAUAAAUUCACG CCUUACGUGAAUUUA 3165 887358 1683990. UUAUUCUdTdT UAAGGdTdT UUCU 1 AD- A- 257 CAAAGGUCACAA A-1683993.1 258 GAGGAAAUUGUGAC CAAAGGUCACAAUUU 3166 887359 1683992. UUUCCUCdTdT CUUUGdTdT CCUC 1 AD- A- 259 UCACAAUUUCCU A-1683995.1 260 UUCCUUGAGGAAAU UCACAAUUUCCUCAA 3167 887360 1683994. CAAGGAAdTdT UGUGAdTdT GGAA 1 AD- A- 261 CCUCAAGGAAAA A-1683997.1 262 UUUAUCUUUUUCCU CCUCAAGGAAAAAGA 3168 887361 1683996. AGAUAAAdTdT UGAGGdTdT UAAA 1 AD- A- 263 GCUUCAUUGUCC A-1683999.1 264 AUCAUGAGGACAAU GCUUCAUUGUCCUCA 3169 887362 1683998. UCAUGAUdTdT GAAGCdTdT UGAU 1 AD- A- 265 CUUCAUUGUCCU A-1684001.1 266 GAUCAUGAGGACAA CUUCAUUGUCCUCAU 3170 887363 1684000. CAUGAUCdTdT UGAAGdTdT GAUC 1 AD- A- 267 UGCAGACAAGAU A-1684003.1 268 AGUGAAGAUCUUGU UGCAGACAAGAUCUU 3171 887364 1684002. CUUCACUdTdT CUGCAdTdT CACU 1 AD- A- 269 CAGACAAGAUCU A-1684005.1 270 UAAGUGAAGAUCUU CAGACAAGAUCUUCA 3172 887365 1684004. UCACUUAdTdT GUCUGdTdT CUUA 1 AD- A- 271 AGACAAGAUCUU A-1684007.1 272 GUAAGUGAAGAUCU AGACAAGAUCUUCAC 3173 887366 1684006. CACUUACdTdT UGUCUdTdT UUAC 1 AD- A- 273 GACAAGAUCUUC A-1684009.1 274 UGUAAGUGAAGAUC GACAAGAUCUUCACU 3174 887367 1684008. ACUUACAdTdT UUGUCdTdT UACA 1 AD- A- 275 ACAAGAUCUUCA A-1684011.1 276 AUGUAAGUGAAGAU ACAAGAUCUUCACUU 3175 887368 1684010. CUUACAUdTdT CUUGUdTdT ACAU 1 AD- A- 277 CAAGAUCUUCAC A-1684013.1 278 GAUGUAAGUGAAGA CAAGAUCUUCACUUA 3176 887369 1684012. UUACAUCdTdT UCUUGdTdT CAUC 1 AD- A- 279 AGAUCUUCACUU A-1684015.1 280 AAGAUGUAAGUGAA AGAUCUUCACUUACA 3177 887370 1684014. ACAUCUUdTdT GAUCUdTdT UCUU 1 AD- A- 281 GAUCUUCACUUA A-1684017.1 282 GAAGAUGUAAGUGA GAUCUUCACUUACAU 3178 887371 1684016. CAUCUUCdTdT AGAUCdTdT CUUC 1 AD- A- 283 UCUUCACUUACA A-1684019.1 284 AUGAAGAUGUAAGU UCUUCACUUACAUCU 3179 887372 1684018. UCUUCAUdTdT GAAGAdTdT UCAU 1 AD- A- 285 CUUCACUUACAU A-1684021.1 286 AAUGAAGAUGUAAG CUUCACUUACAUCUU 3180 887373 1684020. CUUCAUUdTdT UGAAGdTdT CAUU 1 AD- A- 287 UUCACUUACAUC A-1684023.1 288 GAAUGAAGAUGUAA UUCACUUACAUCUUC 3181 887374 1684022. UUCAUUCdTdT GUGAAdTdT AUUC 1 AD- A- 289 UCACUUACAUCU A-1684025.1 290 AGAAUGAAGAUGUA UCACUUACAUCUUCA 3182 887375 1684024. UCAUUCUdTdT AGUGAdTdT UUCU 1 AD- A- 291 CACUUACAUCUU A-1684027.1 292 CAGAAUGAAGAUGU CACUUACAUCUUCAU 3183 887376 1684026. CAUUCUGdTdT AAGUGdTdT UCUG 1 AD- A- 293 CUUACAUCUUCA A-1684029.1 294 UCCAGAAUGAAGAU CUUACAUCUUCAUUC 3184 887377 1684028. UUCUGGAdTdT GUAAGdTdT UGGA 1 AD- A- 295 ACAUCUUCAUUC A-1684031.1 296 AUUUCCAGAAUGAA ACAUCUUCAUUCUGG 3185 887378 1684030. UGGAAAUdTdT GAUGUdTdT AAAU 1 AD- A- 297 CAUCUUCAUUCU A-1684033.1 298 CAUUUCCAGAAUGAA CAUCUUCAUUCUGGA 3186 887379 1684032. GGAAAUGdTdT GAUGdTdT AAUG 1 AD- A- 299 UCUUCAUUCUGG A-1684035.1 300 AGCAUUUCCAGAAU UCUUCAUUCUGGAAA 3187 887380 1684034. AAAUGCUdTdT GAAGAdTdT UGCU 1 AD- A- 301 CUUCAUUCUGGA A-1684037.1 302 AAGCAUUUCCAGAAU CUUCAUUCUGGAAAU 3188 887381 1684036. AAUGCUUdTdT GAAGdTdT GCUU 1 AD- A- 303 UCUGGAAAUGCU A-1684039.1 304 UUUUAGAAGCAUUU UCUGGAAAUGCUUCU 3189 887382 1684038. UCUAAAAdTdT CCAGAdTdT AAAA 1 AD- A- 305 GCUGGAUUUCCU A-1684041.1 306 AACAAUUAGGAAAUC GCUGGAUUUCCUAAU 3190 887383 1684040. AAUUGUUdTdT CAGCdTdT UGUU 1 AD- A- 307 CUGGAUUUCCUA A-1684043.1 308 CAACAAUUAGGAAAU CUGGAUUUCCUAAUU 3191 887384 1684042. AUUGUUGdTdT CCAGdTdT GUUG 1 AD- A- 309 CCUCUAAGAGCC A-1684045.1 310 UAGAUAAGGCUCUU CCUCUAAGAGCCUUA 3192 887385 1684044. UUAUCUAdTdT AGAGGdTdT UCUA 1 AD- A- 311 CUCUAAGAGCCU A-1684047.1 312 CUAGAUAAGGCUCU CUCUAAGAGCCUUAU 3193 887386 1684046. UAUCUAGdTdT UAGAGdTdT CUAG 1 AD- A- 313 CUUCCAUCAUGA A-1684049.1 314 AGCACAUUCAUGAU CUUCCAUCAUGAAUG 3194 887387 1684048. AUGUGCUdTdT GGAAGdTdT UGCU 1 AD- A- 315 UUUCCUGCAAGU A-1684051.1 316 GAACUUGACUUGCA UUUCCUGCAAGUCAA 3195 887388 1684050. CAAGUUCdTdT GGAAAdTdT GUUC 1 AD- A- 317 CUGCAAGUCAAG A-1684053.1 318 UUUGGAACUUGACU CUGCAAGUCAAGUUC 3196 887389 1684052. UUCCAAAdTdT UGCAGdTdT CAAA 1 AD- A- 319 AGUCAAGUUCCA A-1684055.1 320 AACGAUUUGGAACU AGUCAAGUUCCAAAU 3197 887390 1684054. AAUCGUUdTdT UGACUdTdT CGUU 1 AD- A- 321 ACUUGGUUACCU A-1684057.1 322 CAGAGAUAGGUAACC ACUUGGUUACCUAUC 3198 887391 1684056. AUCUCUGdTdT AAGUdTdT UCUG 1 AD- A- 323 CUUGGUUACCUA A-1684059.1 324 GCAGAGAUAGGUAA CUUGGUUACCUAUCU 3199 887392 1684058. UCUCUGCdTdT CCAAGdTdT CUGC 1 AD- A- 325 GGUUACCUAUCU A-1684061.1 326 GAAGCAGAGAUAGG GGUUACCUAUCUCUG 3200 887393 1684060. CUGCUUCdTdT UAACCdTdT CUUC 1 AD- A- 327 GUUACCUAUCUC A-1684063.1 328 UGAAGCAGAGAUAG GUUACCUAUCUCUGC 3201 887394 1684062. UGCUUCAdTdT GUAACdTdT UUCA 1 AD- A- 329 UUACCUAUCUCU A-1684065.1 330 UUGAAGCAGAGAUA UUACCUAUCUCUGCU 3202 887395 1684064. GCUUCAAdTdT GGUAAdTdT UCAA 1 AD- A- 331 UACCUAUCUCUG A-1684067.1 332 CUUGAAGCAGAGAU UACCUAUCUCUGCUU 3203 887396 1684066. CUUCAAGdTdT AGGUAdTdT CAAG 1 AD- A- 333 ACCUAUCUCUGC A-1684069.1 334 ACUUGAAGCAGAGA ACCUAUCUCUGCUUC 3204 887397 1684068. UUCAAGUdTdT UAGGUdTdT AAGU 1 AD- A- 335 CCUAUCUCUGCU A-1684071.1 336 AACUUGAAGCAGAG CCUAUCUCUGCUUCA 3205 887398 1684070. UCAAGUUdTdT AUAGGdTdT AGUU 1 AD- A- 337 CUAUCUCUGCUU A-1684073.1 338 CAACUUGAAGCAGAG CUAUCUCUGCUUCAA 3206 887399 1684072. CAAGUUGdTdT AUAGdTdT GUUG 1 AD- A- 339 AUCUCUGCUUCA A-1684075.1 340 UGCAACUUGAAGCA AUCUCUGCUUCAAGU 3207 887400 1684074. AGUUGCAdTdT GAGAUdTdT UGCA 1 AD- A- 341 UCUCUGCUUCAA A-1684077.1 342 UUGCAACUUGAAGC UCUCUGCUUCAAGUU 3208 887401 1684076. GUUGCAAdTdT AGAGAdTdT GCAA 1 AD- A- 343 CUCUGCUUCAAG A-1684079.1 344 GUUGCAACUUGAAG CUCUGCUUCAAGUUG 3209 887402 1684078. UUGCAACdTdT CAGAGdTdT CAAC 1 AD- A- 345 UCUGCUUCAAGU A-1684081.1 346 AGUUGCAACUUGAA UCUGCUUCAAGUUGC 3210 887403 1684080. UGCAACUdTdT GCAGAdTdT AACU 1 AD- A- 347 UAUCAUCUUUGG A-1684083.1 348 GAAUGACCCAAAGAU UAUCAUCUUUGGGUC 3211 887404 1684082. GUCAUUCdTdT GAUAdTdT AUUC 1 AD- A- 349 AUCAUCUUUGGG A-1684085.1 350 AGAAUGACCCAAAGA AUCAUCUUUGGGUCA 3212 887405 1684084. UCAUUCUdTdT UGAUdTdT UUCU 1 AD- A- 351 UCAUCUUUGGG A-1684087.1 352 AAGAAUGACCCAAAG UCAUCUUUGGGUCAU 3213 887406 1684086. UCAUUCUUdTdT AUGAdTdT UCUU 1 AD- A- 353 CAUCUUUGGGUC A-1684089.1 354 GAAGAAUGACCCAAA CAUCUUUGGGUCAUU 3214 887407 1684088. AUUCUUCdTdT GAUGdTdT CUUC 1 AD- A- 355 CUUUGGGUCAU A-1684091.1 356 AGUGAAGAAUGACCC CUUUGGGUCAUUCUU 3215 887408 1684090. UCUUCACUdTdT AAAGdTdT CACU 1 AD- A- 357 UUGGGUCAUUC A-1684093.1 358 AAAGUGAAGAAUGA UUGGGUCAUUCUUCA 3216 887409 1684092. UUCACUUUdTdT CCCAAdTdT CUUU 1 AD- A- 359 UGGGUCAUUCU A-1684095.1 360 CAAAGUGAAGAAUG UGGGUCAUUCUUCAC 3217 887410 1684094. UCACUUUGdTdT ACCCAdTdT UUUG 1 AD- A- 361 GGGUCAUUCUUC A-1684097.1 362 UCAAAGUGAAGAAU GGGUCAUUCUUCACU 3218 887411 1684096. ACUUUGAdTdT GACCCdTdT UUGA 1 AD- A- 363 GGUCAUUCUUCA A-1684099.1 364 UUCAAAGUGAAGAA GGUCAUUCUUCACUU 3219 887412 1684098. CUUUGAAdTdT UGACCdTdT UGAA 1 AD- A- 365 GUCAUUCUUCAC A-1684101.1 366 GUUCAAAGUGAAGA GUCAUUCUUCACUUU 3220 887413 1684100. UUUGAACdTdT AUGACdTdT GAAC 1 AD- A- 367 CAUUCUUCACUU A-1684103.1 368 AAGUUCAAAGUGAA CAUUCUUCACUUUGA 3221 887414 1684102. UGAACUUdTdT GAAUGdTdT ACUU 1 AD- A- 369 UCACUUUGAACU A-1684105.1 370 AUGAACAAGUUCAAA UCACUUUGAACUUGU 3222 887415 1684104. UGUUCAUdTdT GUGAdTdT UCAU 1 AD- A- 371 CUUGUUCAUUG A-1684107.1 372 GAUGACACCAAUGAA CUUGUUCAUUGGUG 3223 887416 1684106. GUGUCAUCdTdT CAAGdTdT UCAUC 1 AD- A- 373 GUGUCAUCAUAG A-1684109.1 374 AAAUUAUCUAUGAU GUGUCAUCAUAGAUA 3224 887417 1684108. AUAAUUUdTdT GACACdTdT AUUU 1 AD- A- 375 UGUCAUCAUAGA A-1684111.1 376 GAAAUUAUCUAUGA UGUCAUCAUAGAUAA 3225 887418 1684110. UAAUUUCdTdT UGACAdTdT UUUC 1 AD- A- 377 GAGGUCAAGACA A-1684113.1 378 AUAAAGAUGUCUUG GAGGUCAAGACAUCU 3226 887419 1684112. UCUUUAUdTdT ACCUCdTdT UUAU 1 AD- A- 379 AGGUCAAGACAU A-1684115.1 380 CAUAAAGAUGUCUU AGGUCAAGACAUCUU 3227 887420 1684114. CUUUAUGdTdT GACCUdTdT UAUG 1 AD- A- 381 GGUCAAGACAUC A-1684117.1 382 UCAUAAAGAUGUCU GGUCAAGACAUCUUU 3228 887421 1684116. UUUAUGAdTdT UGACCdTdT AUGA 1 AD- A- 383 CCACAAAAGCCAA A-1684119.1 384 GAGGAAUUGGCUUU CCACAAAAGCCAAUUC 3229 887422 1684118. UUCCUCdTdT UGUGGdTdT cue 1 AD- A- 385 GACCUAGUGACA A-1684121.1 386 CUUGAUUUGUCACU GACCUAGUGACAAAU 3230 887423 1684120. AAUCAAGdTdT AGGUCdTdT CAAG 1 AD- A- 387 GUAUCAUGGUUC A-1684123.1 388 CAGAUAAGAACCAUG GUAUCAUGGUUCUUA 3231 887424 1684122. UUAUCUGdTdT AUACdTdT UCUG 1 AD- A- 389 UAUCAUGGUUCU A-1684125.1 390 ACAGAUAAGAACCAU UAUCAUGGUUCUUAU 3232 887425 1684124. UAUCUGUdTdT GAUAdTdT CUGU 1 AD- A- 391 UCAUGGUUCUUA A-1684127.1 392 AGACAGAUAAGAACC UCAUGGUUCUUAUCU 3233 887426 1684126. UCUGUCUdTdT AUGAdTdT GUCU 1 AD- A- 393 CAUGGUUCUUAU A-1684129.1 394 GAGACAGAUAAGAAC CAUGGUUCUUAUCUG 3234 887427 1684128. CUGUCUCdTdT CAUGdTdT UCUC 1 AD- A- 395 AUGGUUCUUAUC A-1684131.1 396 UGAGACAGAUAAGA AUGGUUCUUAUCUGU 3235 887428 1684130. UGUCUCAdTdT ACCAUdTdT CUCA 1 AD- A- 397 UGGUUCUUAUC A-1684133.1 398 UUGAGACAGAUAAG UGGUUCUUAUCUGUC 3236 887429 1684132. UGUCUCAAdTdT AACCAdTdT UCAA 1 AD- A- 399 GGUUCUUAUCU A-1684135.1 400 GUUGAGACAGAUAA GGUUCUUAUCUGUCU 3237 887430 1684134. GUCUCAACdTdT GAACCdTdT CAAC 1 AD- A- 401 GUUCUUAUCUG A-1684137.1 402 UGUUGAGACAGAUA GUUCUUAUCUGUCUC 3238 887431 1684136. UCUCAACAdTdT AGAACdTdT AACA 1 AD- A- 403 UCUUAUCUGUCU A-1684139.1 404 CAUGUUGAGACAGA UCUUAUCUGUCUCAA 3239 887432 1684138. CAACAUGdTdT UAAGAdTdT CAUG 1 AD- A- 405 AUCUGUCUCAAC A-1684141.1 406 UUACCAUGUUGAGA AUCUGUCUCAACAUG 3240 887433 1684140. AUGGUAAdTdT CAGAUdTdT GUAA 1 AD- A- 407 UCUGUCUCAACA A-1684143.1 408 GUUACCAUGUUGAG UCUGUCUCAACAUGG 3241 887434 1684142. UGGUAACdTdT ACAGAdTdT UAAC 1 AD- A- 409 CUGUCUCAACAU A-1684145.1 410 GGUUACCAUGUUGA CUGUCUCAACAUGGU 3242 887435 1684144. GGUAACCdTdT GACAGdTdT AACC 1 AD- A- 411 UCCUGGUCAUGU A-1684147.1 412 UAGAUGAACAUGACC UCCUGGUCAUGUUCA 3243 887436 1684146. UCAUCUAdTdT AGGAdTdT UCUA 1 AD- A- 413 AGUUCAUCCUGG A-1684149.1 414 UGAACUUCCAGGAU AGUUCAUCCUGGAAG 3244 887437 1684148. AAGUUCAdTdT GAACUdTdT UUCA 1 AD- A- 415 CCAUCUGUUGGA A-1684151.1 416 AGAAUAUUCCAACAG CCAUCUGUUGGAAUA 3245 887438 1684150. AUAUUCUdTdT AUGGdTdT UUCU 1 AD- A- 417 CAUCUGUUGGAA A-1684153.1 418 UAGAAUAUUCCAACA CAUCUGUUGGAAUAU 3246 887439 1684152. UAUUCUAdTdT GAUGdTdT UCUA 1 AD- A- 419 UCUGUUGGAAU A-1684155.1 420 AGUAGAAUAUUCCA UCUGUUGGAAUAUUC 3247 887440 1684154. AUUCUACUdTdT ACAGAdTdT UACU 1 AD- A- 421 CAUACUGGAGAA A-1684157.1 422 ACUAAAAUUCUCCAG CAUACUGGAGAAUUU 3248 887441 1684156. UUUUAGUdTdT UAUGdTdT UAGU 1 AD- A- 423 CUCCUCUUCUCA A-1684159.1 424 UUUGCUAUGAGAAG CUCCUCUUCUCAUAG 3249 887442 1684158. UAGCAAAdTdT AGGAGdTdT CAAA 1 AD- A- 425 UCCUCUUCUCAU A-1684161.1 426 UUUUGCUAUGAGAA UCCUCUUCUCAUAGC 3250 887443 1684160. AGCAAAAdTdT GAGGAdTdT AAAA 1 AD- A- 427 CCUCUUCUCAUA A-1684163.1 428 GUUUUGCUAUGAGA CCUCUUCUCAUAGCA 3251 887444 1684162. GCAAAACdTdT AGAGGdTdT AAAC 1 AD- A- 429 CUCUUCUCAUAG A-1684165.1 430 GGUUUUGCUAUGAG CUCUUCUCAUAGCAA 3252 887445 1684164. CAAAACCdTdT AAGAGdTdT AACC 1 AD- A- 431 GAUCCAUUGUCU A-1684167.1 432 GAUGUCAAGACAAU GAUCCAUUGUCUUGA 3253 887446 1684166. UGACAUCdTdT GGAUCdTdT CAUC 1 AD- A- 433 AUCCAUUGUCUU A-1684169.1 434 AGAUGUCAAGACAA AUCCAUUGUCUUGAC 3254 887447 1684168. GACAUCUdTdT UGGAUdTdT AUCU 1 AD- A- 435 UCCAUUGUCUUG A-1684171.1 436 AAGAUGUCAAGACAA UCCAUUGUCUUGACA 3255 887448 1684170. ACAUCUUdTdT UGGAdTdT UCUU 1 AD- A- 437 CAUUGUCUUGAC A-1684173.1 438 AUAAGAUGUCAAGA CAUUGUCUUGACAUC 3256 887449 1684172. AUCUUAUdTdT CAAUGdTdT UUAU 1 AD- A- 439 UUGUCUUGACAU A-1684175.1 440 AAAUAAGAUGUCAA UUGUCUUGACAUCUU 3257 887450 1684174. CUUAUUUdTdT GACAAdTdT AUUU 1 AD- A- 441 UGUCUUGACAUC A-1684177.1 442 CAAAUAAGAUGUCAA UGUCUUGACAUCUUA 3258 887451 1684176. UUAUUUGdTdT GACAdTdT UUUG 1 AD- A- 443 GUCUUGACAUCU A-1684179.1 444 GCAAAUAAGAUGUC GUCUUGACAUCUUAU 3259 887452 1684178. UAUUUGCdTdT AAGACdTdT UUGC 1 AD- A- 445 GGAGAUGGAUUC A-1684181.1 446 ACGAAGAGAAUCCAU GGAGAUGGAUUCUCU 3260 887453 1684180. UCUUCGUdTdT CUCCdTdT UCGU 1 AD- A- 447 GAGAUGGAUUCU A-1684183.1 448 AACGAAGAGAAUCCA GAGAUGGAUUCUCUU 3261 887454 1684182. CUUCGUUdTdT UCUCdTdT CGUU 1 AD- A- 449 AGAUGGAUUCUC A-1684185.1 450 GAACGAAGAGAAUCC AGAUGGAUUCUCUUC 3262 887455 1684184. UUCGUUCdTdT AUCUdTdT GUUC 1 AD- A- 451 GAUGGAUUCUCU A-1684187.1 452 UGAACGAAGAGAAU GAUGGAUUCUCUUCG 3263 887456 1684186. UCGUUCAdTdT CCAUCdTdT UUCA 1 AD- A- 453 AUGGAUUCUCUU A-1684189.1 454 GUGAACGAAGAGAA AUGGAUUCUCUUCGU 3264 887457 1684188. CGUUCACdTdT UCCAUdTdT UCAC 1 AD- A- 455 UGGAUUCUCUUC A-1684191.1 456 UGUGAACGAAGAGA UGGAUUCUCUUCGUU 3265 887458 1684190. GUUCACAdTdT AUCCAdTdT CACA 1 AD- A- 457 GGAUUCUCUUCG A-1684193.1 458 CUGUGAACGAAGAG GGAUUCUCUUCGUUC 3266 887459 1684192. UUCACAGdTdT AAUCCdTdT ACAG 1 AD- A- 459 GAUUCUCUUCGU A-1684195.1 460 UCUGUGAACGAAGA GAUUCUCUUCGUUCA 3267 887460 1684194. UCACAGAdTdT GAAUCdTdT CAGA 1 AD- A- 461 UUCUCUUCGUUC A-1684197.1 462 CAUCUGUGAACGAA UUCUCUUCGUUCACA 3268 887461 1684196. ACAGAUGdTdT GAGAAdTdT GAUG 1 AD- A- 463 UCUCUUCGUUCA A-1684199.1 464 CCAUCUGUGAACGAA UCUCUUCGUUCACAG 3269 887462 1684198. CAGAUGGdTdT GAGAdTdT AUGG 1 AD- A- 465 CUCUUCGUUCAC A-1684201.1 466 UCCAUCUGUGAACGA CUCUUCGUUCACAGA 3270 887463 1684200. AGAUGGAdTdT AGAGdTdT UGGA 1 AD- A- 467 UCUUCGUUCACA A-1684203.1 468 UUCCAUCUGUGAAC UCUUCGUUCACAGAU 3271 887464 1684202. GAUGGAAdTdT GAAGAdTdT GGAA 1 AD- A- 469 AGGUUCAUGUCU A-1684205.1 470 GAUUUGCAGACAUG AGGUUCAUGUCUGCA 3272 887465 1684204. GCAAAUCdTdT AACCUdTdT AAUC 1 AD- A- 471 UCUGCAAAUCCU A-1684207.1 472 CUUUGGAAGGAUUU UCUGCAAAUCCUUCC 3273 887466 1684206. UCCAAAGdTdT GCAGAdTdT AAAG 1 AD- A- 473 CUGCAAAUCCUU A-1684209.1 474 ACUUUGGAAGGAUU CUGCAAAUCCUUCCA 3274 887467 1684208. CCAAAGUdTdT UGCAGdTdT AAGU 1 AD- A- 475 GUGUCUGCUACU A-1684211.1 476 GAAUGACAGUAGCA GUGUCUGCUACUGUC 3275 887468 1684210. GUCAUUCdTdT GACACdTdT AUUC 1 AD- A- 477 UGUCUGCUACUG A-1684213.1 478 UGAAUGACAGUAGC UGUCUGCUACUGUCA 3276 887469 1684212. UCAUUCAdTdT AGACAdTdT UUCA 1 AD- A- 479 GUCUGCUACUGU A-1684215.1 480 CUGAAUGACAGUAG GUCUGCUACUGUCAU 3277 887470 1684214. CAUUCAGdTdT CAGACdTdT UCAG 1 AD- A- 481 ACCGCUUAAGGC A-1684217.1 482 ACAUUUUGCCUUAA ACCGCUUAAGGCAAA 3278 887471 1684216. AAAAUGUdTdT GCGGUdTdT AUGU 1 AD- A- 483 CCGCUUAAGGCA A-1684219.1 484 GACAUUUUGCCUUA CCGCUUAAGGCAAAA 3279 887472 1684218. AAAUGUCdTdT AGCGGdTdT UGUC 1 AD- A- 485 UCUCCACCUUCA A-1684221.1 486 UAUCAUAUGAAGGU UCUCCACCUUCAUAU 3280 887473 1684220. UAUGAUAdTdT GGAGAdTdT GAUA 1 AD- A- 487 UGCCAAAAUCCU A-1684223.1 488 GAUAAAAAGGAUUU UGCCAAAAUCCUUUU 3281 887474 1684222. UUUUAUCdTdT UGGCAdTdT UAUC 1 AD- A- 489 GCCAAAAUCCUU A-1684225.1 490 UGAUAAAAAGGAUU GCCAAAAUCCUUUUU 3282 887475 1684224. UUUAUCAdTdT UUGGCdTdT AUCA 1 AD- A- 491 UCGUAAGAGAAC A-1684227.1 492 CUACAGAGUUCUCU UCGUAAGAGAACUCU 3283 887476 1684226. UCUGUAGdTdT UACGAdTdT GUAG 1 AD- A- 493 UCUGCCUUGUCA A-1684229.1 494 GAAAAGAUGACAAG UCUGCCUUGUCAUCU 3284 887477 1684228. UCUUUUCdTdT GCAGAdTdT UUUC 1 AD- A- 495 CUGCCUUGUCAU A-1684231.1 496 UGAAAAGAUGACAA CUGCCUUGUCAUCUU 3285 887478 1684230. CUUUUCAdTdT GGCAGdTdT UUCA 1 AD- A- 497 UGCCUUGUCAUC A-1684233.1 498 GUGAAAAGAUGACA UGCCUUGUCAUCUUU 3286 887479 1684232. UUUUCACdTdT AGGCAdTdT UCAC 1 AD- A- 499 GCCUUGUCAUCU A-1684235.1 500 UGUGAAAAGAUGAC GCCUUGUCAUCUUUU 3287 887480 1684234. UUUCACAdTdT AAGGCdTdT CACA 1 AD- A- 501 CCUUGUCAUCUU A-1684237.1 502 CUGUGAAAAGAUGA CCUUGUCAUCUUUUC 3288 887481 1684236. UUCACAGdTdT CAAGGdTdT ACAG 1 AD- A- 503 CAUCUUUUCACA A-1684239.1 504 ACAAUCCUGUGAAAA CAUCUUUUCACAGGA 3289 887482 1684238. GGAUUGUdTdT GAUGdTdT UUGU 1 AD- A- 505 CCCAUGUAAAUA A-1684241.1 506 UGUUGUUUAUUUAC CCCAUGUAAAUAAAC 3290 887483 1684240. AACAACAdTdT AUGGGdTdT AACA 1 AD- A- 507 CAUUCAUCUUGA A-1684243.1 508 AUGUGAGUCAAGAU CAUUCAUCUUGACUC 3291 887484 1684242. CUCACAUdTdT GAAUGdTdT ACAU 1 AD- A- 509 ACAUAUUACACU A-1684245.1 510 UUUGAGGAGUGUAA ACAUAUUACACUCCU 3292 887485 1684244. CCUCAAAdTdT UAUGUdTdT CAAA 1 AD- A- 511 CAUAUUACACUC A-1684247.1 512 UUUUGAGGAGUGUA CAUAUUACACUCCUC 3293 887486 1684246. CUCAAAAdTdT AUAUGdTdT AAAA 1 AD- A- 513 UGCCCAAAAUAC A-1684249.1 514 AUUAUCAGUAUUUU UGCCCAAAAUACUGA 3294 887487 1684248. UGAUAAUdTdT GGGCAdTdT UAAU 1 AD- A- 515 GCCCAAAAUACU A-1684251.1 516 UAUUAUCAGUAUUU GCCCAAAAUACUGAU 3295 887488 1684250. GAUAAUAdTdT UGGGCdTdT AAUA 1 AD- A- 517 CUGAUAAUAGUC A-1684253.1 518 UUUAAGAGACUAUU CUGAUAAUAGUCUCU 3296 887489 1684252. UCUUAAAdTdT AUCAGdTdT UAAA 1 AD- A- 519 GUCAAAUUUUCC A-1684255.1 520 GAAAGCAGGAAAAU GUCAAAUUUUCCUGC 3297 887490 1684254. UGCUUUCdTdT UUGACdTdT UUUC 1 AD- A- 521 UCAAAUUUUCCU A-1684257.1 522 AGAAAGCAGGAAAAU UCAAAUUUUCCUGCU 3298 887491 1684256. GCUUUCUdTdT UUGAdTdT UUCU 1 AD- A- 523 CAAAUUUUCCUG A-1684259.1 524 AAGAAAGCAGGAAAA CAAAUUUUCCUGCUU 3299 887492 1684258. CUUUCUUdTdT UUUGdTdT UCUU 1 AD- A- 525 AUUGUUUAGUC A-1684261.1 526 GAAAGGAUGACUAA AUUGUUUAGUCAUCC 3300 887493 1684260. AUCCUUUCdTdT ACAAUdTdT UUUC 1 AD- A- 527 GCAUCACUUGUA A-1684263.1 528 GAUUGUAUACAAGU GCAUCACUUGUAUAC 3301 887494 1684262. UACAAUCdTdT GAUGCdTdT AAUC 1 AD- A- 529 CACCAACUUACU A-1684265.1 530 UUAGGAAAGUAAGU CACCAACUUACUUUC 3302 887495 1684264. UUCCUAAdTdT UGGUGdTdT CUAA 1 AD- A- 531 ACCAACUUACUU A-1684267.1 532 UUUAGGAAAGUAAG ACCAACUUACUUUCC 3303 887496 1684266. UCCUAAAdTdT UUGGUdTdT UAAA 1 AD- A- 533 CCAACUUACUUU A-1684269.1 534 AUUUAGGAAAGUAA CCAACUUACUUUCCU 3304 887497 1684268. CCUAAAUdTdT GUUGGdTdT AAAU 1 AD- A- 535 CAACUUACUUUC A-1684271.1 536 AAUUUAGGAAAGUA CAACUUACUUUCCUA 3305 887498 1684270. CUAAAUUdTdT AGUUGdTdT AAUU 1 AD- A- 537 AGGAAGAUGUCA A-1684273.1 538 GAGAAGGUGACAUC AGGAAGAUGUCACCU 3306 887499 1684272. CCUUCUCdTdT UUCCUdTdT UCUC 1 AD- A- 539 GAAGAUGUCACC A-1684275.1 540 AGGAGAAGGUGACA GAAGAUGUCACCUUC 3307 887500 1684274. UUCUCCUdTdT UCUUCdTdT UCCU 1 AD- A- 541 AGAUGUCACCUU A-1684277.1 542 UAAGGAGAAGGUGA AGAUGUCACCUUCUC 3308 887501 1684276. CUCCUUAdTdT CAUCUdTdT CUUA 1 AD- A- 543 GAUGUCACCUUC A-1684279.1 544 UUAAGGAGAAGGUG GAUGUCACCUUCUCC 3309 887502 1684278. UCCUUAAdTdT ACAUCdTdT UUAA 1 AD- A- 545 AUGUCACCUUCU A-1684281.1 546 UUUAAGGAGAAGGU AUGUCACCUUCUCCU 3310 887503 1684280. CCUUAAAdTdT GACAUdTdT UAAA 1 AD- A- 547 UGUCACCUUCUC A-1684283.1 548 UUUUAAGGAGAAGG UGUCACCUUCUCCUU 3311 887504 1684282. CUUAAAAdTdT UGACAdTdT AAAA 1 AD- A- 549 GUCACCUUCUCC A-1684285.1 550 AUUUUAAGGAGAAG GUCACCUUCUCCUUA 3312 887505 1684284. UUAAAAUdTdT GUGACdTdT AAAU 1 AD- A- 551 UCACCUUCUCCU A-1684287.1 552 AAUUUUAAGGAGAA UCACCUUCUCCUUAA 3313 887506 1684286. UAAAAUUdTdT GGUGAdTdT AAUU 1 AD- A- 553 ACCUUCUCCUUA A-1684289.1 554 AGAAUUUUAAGGAG ACCUUCUCCUUAAAA 3314 887507 1684288. AAAUUCUdTdT AAGGUdTdT UUCU 1 AD- A- 555 CCUUCUCCUUAA A-1684291.1 556 UAGAAUUUUAAGGA CCUUCUCCUUAAAAU 3315 887508 1684290. AAUUCUAdTdT GAAGGdTdT UCUA 1 AD- A- 557 CUUCUCCUUAAA A-1684293.1 558 AUAGAAUUUUAAGG CUUCUCCUUAAAAUU 3316 887509 1684292. AUUCUAUdTdT AGAAGdTdT CUAU 1 AD- A- 559 UGAGAUCUUUCU A-1684295.1 560 UUAUAGAAGAAAGA UGAGAUCUUUCUUCU 3317 887510 1684294. UCUAUAAdTdT UCUCAdTdT AUAA 1 AD- A- 561 GAUCUUUCUUCU A-1684297.1 562 ACUUUAUAGAAGAA GAUCUUUCUUCUAUA 3318 887511 1684296. AUAAAGUdTdT AGAUCdTdT AAGU 1 AD- A- 563 UACCAUCUUAGG A-1684299.1 564 GAAUGAACCUAAGA UACCAUCUUAGGUUC 3319 887512 1684298. UUCAUUCdTdT UGGUAdTdT AUUC 1 AD- A- 565 ACCAUCUUAGGU A-1684301.1 566 UGAAUGAACCUAAG ACCAUCUUAGGUUCA 3320 887513 1684300. UCAUUCAdTdT AUGGUdTdT UUCA 1 AD- A- 567 CCAUCUUAGGUU A-1684303.1 568 AUGAAUGAACCUAA CCAUCUUAGGUUCAU 3321 887514 1684302. CAUUCAUdTdT GAUGGdTdT UCAU 1 AD- A- 569 CAUCUUAGGUUC A-1684305.1 570 GAUGAAUGAACCUA CAUCUUAGGUUCAUU 3322 887515 1684304. AUUCAUCdTdT AGAUGdTdT CAUC 1 AD- A- 571 UCUUAGGUUCAU A-1684307.1 572 AAGAUGAAUGAACC UCUUAGGUUCAUUCA 3323 887516 1684306. UCAUCUUdTdT UAAGAdTdT UCUU 1 AD- A- 573 CUUAGGUUCAUU A-1684309.1 574 UAAGAUGAAUGAAC CUUAGGUUCAUUCAU 3324 887517 1684308. CAUCUUAdTdT CUAAGdTdT CUUA 1 AD- A- 575 UUAGGUUCAUUC A-1684311.1 576 CUAAGAUGAAUGAA UUAGGUUCAUUCAUC 3325 887518 1684310. AUCUUAGdTdT CCUAAdTdT UUAG 1 AD- A- 577 UAGGUUCAUUCA A-1684313.1 578 CCUAAGAUGAAUGA UAGGUUCAUUCAUCU 3326 887519 1684312. UCUUAGGdTdT ACCUAdTdT UAGG 1 AD- A- 579 CUGCAUUAUGAA A-1684315.1 580 GUAAGUAUUCAUAA CUGCAUUAUGAAUAC 3327 887520 1684314. UACUUACdTdT UGCAGdTdT UUAC 1 AD- A- 581 ACACAAUUUCUU A-1684317.1 582 UGCUAAGAAGAAAU ACACAAUUUCUUCUU 3328 887521 1684316. CUUAGCAdTdT UGUGUdTdT AGCA 1 AD- A- 583 GUUCUUUUUCC A-1684319.1 584 AUGAAAUAGGAAAA GUUCUUUUUCCUAUU 3329 887522 1684318. UAUUUCAUdTdT AGAACdTdT UCAU 1 AD- A- 585 UCCUAUUUCAUG A-1684321.1 586 CAUAGUUCAUGAAA UCCUAUUUCAUGAAC 3330 887523 1684320. AACUAUGdTdT UAGGAdTdT UAUG 1 AD- A- 587 CCUAUUUCAUGA A-1684323.1 588 ACAUAGUUCAUGAA CCUAUUUCAUGAACU 3331 887524 1684322. ACUAUGUdTdT AUAGGdTdT AUGU 1 AD- A- 589 AUGUCUACUUGU A-1684325.1 590 AAAAGUCACAAGUAG AUGUCUACUUGUGAC 3332 887525 1684324. GACUUUUdTdT ACAUdTdT UUUU 1 AD- A- 591 UGUCUACUUGU A-1684327.1 592 AAAAAGUCACAAGUA UGUCUACUUGUGACU 3333 887526 1684326. GACUUUUUdTdT GACAdTdT UUUU 1 AD- A- 593 UCUACUUGUGAC A-1684329.1 594 AUAAAAAGUCACAAG UCUACUUGUGACUUU 3334 887527 1684328. UUUUUAUdTdT UAGAdTdT UUAU 1 AD- A- 595 CUACUUGUGACU A-1684331.1 596 GAUAAAAAGUCACAA CUACUUGUGACUUUU 3335 887528 1684330. UUUUAUCdTdT GUAGdTdT UAUC 1 AD- A- 597 GUUCUAAAUAGC A-1684333.1 598 UGAAAUAGCUAUUU GUUCUAAAUAGCUAU 3336 887529 1684332. UAUUUCAdTdT AGAACdTdT UUCA 1 AD- A- 599 GCUGUUUACAUA A-1684335.1 600 AGAAUCCUAUGUAA GCUGUUUACAUAGGA 3337 887530 1684334. GGAUUCUdTdT ACAGCdTdT UUCU 1 AD- A- 601 GCUCAAAAUGUU A-1684337.1 602 AAACUCAAACAUUUU GCUCAAAAUGUUUGA 3338 887531 1684336. UGAGUUUdTdT GAGCdTdT GUUU 1

TABLE 2B Exemplary Human SCN9A Unmodified Single Strands and Duplex Sequences. Column 1 indicates duplex name. Column 2 indicates the sense sequence name. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the unmodified sequence of a sense strand suitable for use in a duplex described herein. Column 5 provides the position in the target mRNA (NM_002977.3) of the sense strand of Column 4. Column 6 indicates the antisense sequence name. Column 7 indicates the sequence ID for the sequence of column 8. Column 8 provides the sequence of an antisense strand suitable for use in a duplex described herein, without specifying chemical modifications. Column 9 indicates the position in the target mRNA (NM_002977.3) that is complementary to the antisense strand of Column 8. Anti Seq ID Sense Seq ID mRNA target sense NO: mRNA target Duplex sequence NO: Sense sequence range in sequence (anti antisense sequence range in Name name (sense) (5′-3′) NM_002977.3 name sense) (5′-3′) NM_002977.3 AD-887232 A-1683738.1  603 UCACAAAACAGUCUCU  342-360 A-  604 GCAAGAGACUGU  342-360 UGC 1683739.1 UUUGUGA AD-887233 A-1683740.1  605 GGAAAACAAUCUUCCG  579-597 A-  606 AAACGGAAGAUU  579-597 UUU 1683741.1 GUUUUCC AD-887234 A-1683742.1  607 GAAAACAAUCUUCCGU  580-598 A-  608 GAAACGGAAGAU  580-598 UUC 1683743.1 UGUUUUC AD-887235 A-1683744.1  609 AAAACAAUCUUCCGUU  581-599 A-  610 UGAAACGGAAGA  581-599 UCA 1683745.1 UUGUUUU AD-887236 A-1683746.1  611 AAACAAUCUUCCGUUU  582-600 A-  612 UUGAAACGGAAG  582-600 CAA 1683747.1 AUUGUUU AD-887237 A-1683748.1  613 AACAAUCUUCCGUUUC  583-601 A-  614 AUUGAAACGGAA  583-601 AAU 1683749.1 GAUUGUU AD-887238 A-1683750.1  615 CAAUCUUCCGUUUCAA  585-603 A-  616 GCAUUGAAACGG  585-603 UGC 1683751.1 AAGAUUG AD-887239 A-1683752.1  617 CCUGCUUUAUAUAUGC  608-626 A-  618 AAAGCAUAUAUA  608-626 UUU 1683753.1 AAGCAGG AD-887240 A-1683754.1  619 CUGCUUUAUAUAUGC  609-627 A-  620 GAAAGCAUAUAU  609-627 UUUC 1683755.1 AAAGCAG AD-887241 A-1683756.1  621 UAUGCUUUCUCCUUUC  619-637 A-  622 ACUGAAAGGAGA  619-637 AGU 1683757.1 AAGCAUA AD-887242 A-1683758.1  623 AUGCUUUCUCCUUUCA  620-638 A-  624 GACUGAAAGGAG  620-638 GUC 1683759.1 AAAGCAU AD-887243 A-1683760.1  625 UGCUUUCUCCUUUCAG  621-639 A-  626 GGACUGAAAGGA  621-639 UCC 1683761.1 GAAAGCA AD-887244 A-1683762.1  627 CUUUCUCCUUUCAGUC  623-641 A-  628 GAGGACUGAAAG  623-641 CUC 1683763.1 GAGAAAG AD-887245 A-1683764.1  629 UCUCCUUUCAGUCCUC  626-644 A-  630 UUAGAGGACUGA  626-644 UAA 1683765.1 AAGGAGA AD-887246 A-1683766.1  631 CUCCUUUCAGUCCUCU  627-645 A-  632 CUUAGAGGACUG  627-645 AAG 1683767.1 AAAGGAG AD-887247 A-1683768.1  633 UCCUUUCAGUCCUCUA  628-646 A-  634 UCUUAGAGGACU  628-646 AGA 1683769.1 GAAAGGA AD-887248 A-1683770.1  635 CCUUUCAGUCCUCUAA  629-647 A-  636 UUCUUAGAGGAC  629-647 GAA 1683771.1 UGAAAGG AD-887249 A-1683772.1  637 CUUUCAGUCCUCUAAG  630-648 A-  638 CUUCUUAGAGGA  630-648 AAG 1683773.1 CUGAAAG AD-887250 A-1683774.1  639 AGUCCUCUAAGAAGAA  635-653 A-  640 AUAUUCUUCUUA  635-653 UAU 1683775.1 GAGGACU AD-887251 A-1683776.1  641 UCCUCUAAGAAGAAUA  637-655 A-  642 AGAUAUUCUUCU  637-655 UCU 1683777.1 UAGAGGA AD-887252 A-1683778.1  643 CCUCUAAGAAGAAUAU  638-656 A-  644 UAGAUAUUCUUC  638-656 CUA 1683779.1 UUAGAGG AD-887253 A-1683780.1  645 CUCUAAGAAGAAUAUC  639-657 A-  646 AUAGAUAUUCUU  639-657 UAU 1683781.1 CUUAGAG AD-887254 A-1683782.1  647 AUUUUAGUACACUCCU  662-680 A-  648 AUAAGGAGUGUA  662-680 UAU 1683783.1 CUAAAAU AD-887255 A-1683784.1  649 UAGUACACUCCUUAUU  666-684 A-  650 CUGAAUAAGGAG  666-684 CAG 1683785.1 UGUACUA AD-887256 A-1683786.1  651 AGUACACUCCUUAUUC  667-685 A-  652 GCUGAAUAAGGA  667-685 AGC 1683787.1 GUGUACU AD-887257 A-1683788.1  653 CCUUAUUCAGCAUGCU  675-693 A-  654 AUGAGCAUGCUG  675-693 CAU 1683789.1 AAUAAGG AD-887258 A-1683790.1  655 UCAUCAUGUGCACUAU  690-708 A-  656 AGAAUAGUGCAC  690-708 UCU 1683791.1 AUGAUGA AD-887259 A-1683792.1  657 CAUCAUGUGCACUAUU  691-709 A-  658 CAGAAUAGUGCA  691-709 CUG 1683793.1 CAUGAUG AD-887260 A-1683794.1  659 UGUCGAGUACACUUU  760-778 A-  660 AGUAAAAGUGUA  760-778 UACU 1683795.1 CUCGACA AD-887261 A-1683796.1  661 GUCGAGUACACUUUUA  761-779 A-  662 CAGUAAAAGUGU  761-779 CUG 1683797.1 ACUCGAC AD-887262 A-1683798.1  663 CUUCUGUGUAGGAGA  823-841 A-  664 GAAUUCUCCUAC  823-841 AUUC 1683799.1 ACAGAAG AD-887263 A-1683800.1  665 UAGGAGAAUUCACUU  831-849 A-  666 AGAAAAGUGAAU  831-849 UUCU 1683801.1 UCUCCUA AD-887264 A-1683802.1  667 AGGAGAAUUCACUUU  832-850 A-  668 AAGAAAAGUGAA  832-850 UCUU 1683803.1 UUCUCCU AD-887265 A-1683804.1  669 GGAGAAUUCACUUUUC  833-851 A-  670 GAAGAAAAGUGA  833-851 UUC 1683805.1 AUUCUCC AD-887266 A-1683806.1  671 GGCAAUGUUUCAGCUC  920-938 A-  672 GAAGAGCUGAAA  920-938 UUC 1683807.1 CAUUGCC AD-887267 A-1683808.1  673 AAUGUUUCAGCUCUUC  923-941 A-  674 UUCGAAGAGCUG  923-941 GAA 1683809.1 AAACAUU AD-887268 A-1683810.1  675 GUUUCAGCUCUUCGAA  926-944 A-  676 AAGUUCGAAGAG  926-944 CUU 1683811.1 CUGAAAC AD-887269 A-1683812.1  677 UCAGCUCUUCGAACUU  929-947 A-  678 UGAAAGUUCGAA  929-947 UCA 1683813.1 GAGCUGA AD-887270 A-1683814.1  679 AGCUCUUCGAACUUUC  931-949 A- 5804 UCUGAAAGUUCG  931-949 AGA 1683815.1 AAGAGCU AD-887271 A-1683816.1 5805 CUCUUCGAACUUUCAG  933-951 A- 5806 ACUCUGAAAGUU  933-951 AGU 1683817.1 CGAAGAG AD-887272 A-1683818.1 5807 CUUCGAACUUUCAGAG  935-953 A- 5808 AUACUCUGAAAG  935-953 UAU 1683819.1 UUCGAAG AD-887273 A-1683820.1 5809 UCCUGACUGUGUUCU 1047-1065 A- 5810 AGACAGAACACAG 1047-1065 GUCU 1683821.1 UCAGGA AD-887274 A-1683822.1 5811 CUGACUGUGUUCUGU 1049-1067 A- 5812 UCAGACAGAACAC 1049-1067 CUGA 1683823.1 AGUCAG AD-887275 A-1683824.1 5813 UGACUGUGUUCUGUC 1050-1068 A-  680 CUCAGACAGAACA 1050-1068 UGAG 1683825.1 CAGUCA AD-887276 A-1683826.1  681 GACUGUGUUCUGUCU 1051-1069 A-  682 ACUCAGACAGAAC 1051-1069 GAGU 1683827.1 ACAGUC AD-887277 A-1683828.1  683 ACUGUGUUCUGUCUG 1052-1070 A-  684 CACUCAGACAGAA 1052-1070 AGUG 1683829.1 CACAGU AD-887278 A-1683830.1  685 CUGUGUUCUGUCUGA 1053-1071 A-  686 ACACUCAGACAGA 1053-1071 GUGU 1683831.1 ACACAG AD-887279 A-1683832.1  687 UGUGUUCUGUCUGAG 1054-1072 A-  688 CACACUCAGACAG 1054-1072 UGUG 1683833.1 AACACA AD-887280 A-1683834.1  689 UGUUCUGUCUGAGUG 1056-1074 A-  690 AACACACUCAGAC 1056-1074 UGUU 1683835.1 AGAACA AD-887281 A-1683836.1  691 GUUCUGUCUGAGUGU 1057-1075 A-  692 AAACACACUCAGA 1057-1075 GUUU 1683837.1 CAGAAC AD-887282 A-1683838.1  693 UUCUGUCUGAGUGUG 1058-1076 A-  694 CAAACACACUCAG 1058-1076 UUUG 1683839.1 ACAGAA AD-887283 A-1683840.1  695 UCUGUCUGAGUGUGU 1059-1077 A-  696 GCAAACACACUCA 1059-1077 UUGC 1683841.1 GACAGA AD-887284 A-1683842.1  697 UGCUCUCCUUUGUGG 1231-1249 A-  698 GAAACCACAAAGG 1231-1249 UUUC 1683843.1 AGAGCA AD-887285 A-1683844.1  699 CUCUCCUUUGUGGUU 1233-1251 A-  700 CUGAAACCACAAA 1233-1251 UCAG 1683845.1   GGAGAG AD-887286 A-1683846.1  701 UCUCCUUUGUGGUUU 1234-1252 A-  702 GCUGAAACCACAA 1234-1252 CAGC 1683847.1 AGGAGA AD-887287 A-1683848.1  703 CUCCUUUGUGGUUUC 1235-1253 A-  704 UGCUGAAACCACA 1235-1253 AGCA 1683849.1 AAGGAG AD-887288 A-1683850.1  705 CGAGCUUUGACACUUU 1323-1341 A-  706 CUGAAAGUGUCA 1323-1341 CAG 1683851.1 AAGCUCG AD-887289 A-1683852.1  707 ACAUGAUCUUCUUUG 1431-1449 A-  708 ACGACAAAGAAGA 1431-1449 UCGU 1683853.1 UCAUGU AD-887290 A-1683854.1  709 CAUGAUCUUCUUUGU 1432-1450 A-  710 UACGACAAAGAA 1432-1450 CGUA 1683855.1 GAUCAUG AD-887291 A-1683856.1  711 GAUCUUCUUUGUCGU 1435-1453 A-  712 CACUACGACAAAG 1435-1453 AGUG 1683857.1 AAGAUC AD-887292 A-1683858.1  713 UCUUCUUUGUCGUAG 1437-1455 A-  714 AUCACUACGACAA 1437-1455 UGAU 1683859.1 AGAAGA AD-887293 A-1683860.1  715 CUUCUUUGUCGUAGU 1438-1456 A-  716 AAUCACUACGACA 1438-1456 GAUU 1683861.1 AAGAAG AD-887294 A-1683862.1  717 UUGUCGUAGUGAUUU 1443-1461 A-  718 AGGAAAAUCACU 1443-1461 UCCU 1683863.1 ACGACAA AD-887295 A-1683864.1  719 GCUCCUUUUAUCUAAU 1464-1482 A-  720 UUUAUUAGAUAA 1464-1482 AAA 1683865.1 AAGGAGC AD-887296 A-1683866.1  721 CUCCUUUUAUCUAAUA 1465-1483 A-  722 GUUUAUUAGAUA 1465-1483 AAC 1683867.1 AAAGGAG AD-887297 A-1683868.1  723 CCUCUCAGAGAGUUCU 1669-1687 A-  724 AGAAGAACUCUC 1669-1687 UCU 1683869.1 UGAGAGG AD-887298 A-1683870.1  725 CUCUCAGAGAGUUCUU 1670-1688 A-  726 CAGAAGAACUCUC 1670-1688 CUG 1683871.1 UGAGAG AD-887299 A-1683872.1  727 UCUCAGAGAGUUCUUC 1671-1689 A-  728 UCAGAAGAACUC 1671-1689 UGA 1683873.1 UCUGAGA AD-887300 A-1683874.1  729 CUCAGAGAGUUCUUCU 1672-1690 A-  730 UUCAGAAGAACU 1672-1690 GAA 1683875.1 CUCUGAG AD-887301 A-1683876.1  731 UCAGAGAGUUCUUCU 1673-1691 A-  732 UUUCAGAAGAAC 1673-1691 GAAA 1683877.1 UCUCUGA AD-887302 A-1683878.1  733 CAGAGAGUUCUUCUGA 1674-1692 A-  734 GUUUCAGAAGAA 1674-1692 AAC 1683879.1 CUCUCUG AD-887303 A-1683880.1  735 GAGAGUUCUUCUGAA 1676-1694 A-  736 AUGUUUCAGAAG 1676-1694 ACAU 1683881.1 AACUCUC AD-887304 A-1683882.1  737 AGAGUUCUUCUGAAAC 1677-1695 A-  738 GAUGUUUCAGAA 1677-1695 AUC 1683883.1 GAACUCU AD-887305 A-1683884.1  739 GAGUUCUUCUGAAACA 1678-1696 A-  740 GGAUGUUUCAGA 1678-1696 UCC 1683885.1 AGAACUC AD-887306 A-1683886.1  741 AGUUCUUCUGAAACAU 1679-1697 A-  742 UGGAUGUUUCAG 1679-1697 CCA 1683887.1 AAGAACU AD-887307 A-1683888.1  743 GUUCUUCUGAAACAUC 1680-1698 A-  744 UUGGAUGUUUCA 1680-1698 CAA 1683889.1 GAAGAAC AD-887308 A-1683890.1  745 UCUUCUGAAACAUCCA 1682-1700 A-  746 GUUUGGAUGUUU 1682-1700 AAC 1683891.1 CAGAAGA AD-887309 A-1683892.1  747 CUUCUGAAACAUCCAA 1683-1701 A-  748 AGUUUGGAUGUU 1683-1701 ACU 1683893.1 UCAGAAG AD-887310 A-1683894.1  749 UCUGAAACAUCCAAAC 1685-1703 A-  750 UCAGUUUGGAUG 1685-1703 UGA 1683895.1 UUUCAGA AD-887311 A-1683896.1  751 UCCAAACUGAGCUCUA 1694-1712 A-  752 UUUUAGAGCUCA 1694-1712 AAA 1683897.1 GUUUGGA AD-887312 A-1683898.1  753 AGGCGUUGUAGUUCC 2300-2318 A-  754 GAUAGGAACUAC 2300-2318 UAUC 1683899.1 AACGCCU AD-887313 A-1683900.1  755 GCGUUGUAGUUCCUA 2302-2320 A-  756 GAGAUAGGAACU 2302-2320 UCUC 1683901.1 ACAACGC AD-887314 A-1683902.1  757 CGUUGUAGUUCCUAU 2303-2321 A-  758 GGAGAUAGGAAC 2303-2321 CUCC 1683903.1 UACAACG AD-887315 A-1683904.1  759 GUUGUAGUUCCUAUC 2304-2322 A-  760 AGGAGAUAGGAA 2304-2322 UCCU 1683905.1 CUACAAC AD-887316 A-1683906.1  761 UUGUAGUUCCUAUCU 2305-2323 A-  762 AAGGAGAUAGGA 2305-2323 CCUU 1683907.1 ACUACAA AD-887317 A-1683908.1  763 UGUAGUUCCUAUCUCC 2306-2324 A-  764 AAAGGAGAUAGG 2306-2324 UUU 1683909.1 AACUACA AD-887318 A-1683910.1  765 GUAGUUCCUAUCUCCU 2307-2325 A-  766 GAAAGGAGAUAG 2307-2325 UUC 1683911.1 GAACUAC AD-887319 A-1683912.1  767 UAGUUCCUAUCUCCUU 2308-2326 A-  768 UGAAAGGAGAUA 2308-2326 UCA 1683913.1 GGAACUA AD-887320 A-1683914.1  769 AGUUCCUAUCUCCUUU 2309-2327 A-  770 CUGAAAGGAGAU 2309-2327 CAG 1683915.1 AGGAACU AD-887321 A-1683916.1  771 GUUCCUAUCUCCUUUC 2310-2328 A-  772 UCUGAAAGGAGA 2310-2328 AGA 1683917.1 UAGGAAC AD-887322 A-1683918.1  773 UUCCUAUCUCCUUUCA 2311-2329 A-  774 CUCUGAAAGGAG 2311-2329 GAG 1683919.1 AUAGGAA AD-887323 A-1683920.1  775 UCCUAUCUCCUUUCAG 2312-2330 A-  776 CCUCUGAAAGGA 2312-2330 AGG 1683921.1 GAUAGGA AD-887324 A-1683922.1  777 UCUCCUUUCAGAGGAU 2317-2335 A-  778 CAUAUCCUCUGA 2317-2335 AUG 1683923.1 AAGGAGA AD-887325 A-1683924.1  779 GCAUAUUAACAAACAC 2379-2397 A-  780 ACAGUGUUUGUU 2379-2397 UGU 1683925.1 AAUAUGC AD-887326 A-1683926.1  781 CUUGAUCUGGAAUUG 2461-2479 A-  782 AGAGCAAUUCCA 2461-2479 CUCU 1683927.1 GAUCAAG AD-887327 A-1683928.1  783 CUCUCCAUAUUGGAUA 2476-2494 A-  784 UUUUAUCCAAUA 2476-2494 AAA 1683929.1 UGGAGAG AD-887328 A-1683930.1  785 UCUCCAUAUUGGAUAA 2477-2495 A-  786 AUUUUAUCCAAU 2477-2495 AAU 1683931.1 AUGGAGA AD-887329 A-1683932.1  787 CUCCAUAUUGGAUAAA 2478-2496 A-  788 AAUUUUAUCCAA 2478-2496 AUU 1683933.1 UAUGGAG AD-887330 A-1683934.1  789 GAUCUUGCAAUUACCA 2537-2555 A-  790 AAAUGGUAAUUG 2537-2555 UUU 1683935.1 CAAGAUC AD-887331 A-1683936.1  791 UUGGUCUUUACUGGA 2639-2657 A-  792 AGAUUCCAGUAA 2639-2657 AUCU 1683937.1 AGACCAA AD-887332 A-1683938.1  793 GGUCUUUACUGGAAU 2641-2659 A-  794 AAAGAUUCCAGU 2641-2659 CUUU 1683939.1 AAAGACC AD-887333 A-1683940.1  795 GUCUUUACUGGAAUC 2642-2660 A-  796 CAAAGAUUCCAG 2642-2660 UUUG 1683941.1 UAAAGAC AD-887334 A-1683942.1  797 GCCUUAUUGUGACUU 2736-2754 A-  798 CUUAAAGUCACA 2736-2754 UAAG 1683943.1 AUAAGGC AD-887335 A-1683944.1  799 GCUCUUUCUAGCAGAU 2764-2782 A-  800 CACAUCUGCUAG 2764-2782 GUG 1683945.1 AAAGAGC AD-887336 A-1683946.1  801 CUCUUUCUAGCAGAUG 2765-2783 A-  802 CCACAUCUGCUAG 2765-2783 UGG 1683947.1 AAAGAG AD-887337 A-1683948.1  803 GUCAGUUCUGCGAUCA 2791-2809 A-  804 GAAUGAUCGCAG 2791-2809 UUC 1683949.1 AACUGAC AD-887338 A-1683950.1  805 UCAGUUCUGCGAUCAU 2792-2810 A-  806 UGAAUGAUCGCA 2792-2810 UCA 1683951.1 GAACUGA AD-887339 A-1683952.1  807 AGUCUUCAAGUUGGCA 2821-2839 A-  808 UUUUGCCAACUU 2821-2839 AAA 1683953.1 GAAGACU AD-887340 A-1683954.1  809 UCUUCAAGUUGGCAAA 2823-2841 A-  810 GAUUUUGCCAAC 2823-2841 AUC 1683955.1 UUGAAGA AD-887341 A-1683956.1  811 CUUCAAGUUGGCAAAA 2824-2842 A-  812 GGAUUUUGCCAA 2824-2842 UCC 1683957.1 CUUGAAG AD-887342 A-1683958.1  813 CCAUCAUCGUCUUCAU 2919-2937 A-  814 AAAAUGAAGACG 2919-2937 UUU 1683959.1 AUGAUGG AD-887343 A-1683960.1  815 CAUCAUCGUCUUCAUU 2920-2938 A-  816 AAAAAUGAAGAC 2920-2938 UUU 1683961.1 GAUGAUG AD-887344 A-1683962.1  817 GCACAUGAACGACUUC 3022-3040 A-  818 GAAGAAGUCGUU 3022-3040 UUC 1683963.1 CAUGUGC AD-887345 A-1683964.1  819 CACAUGAACGACUUCU 3023-3041 A-  820 GGAAGAAGUCGU 3023-3041 UCC 1683965.1 UCAUGUG AD-887346 A-1683966.1  821 ACAUGAACGACUUCUU 3024-3042 A-  822 UGGAAGAAGUCG 3024-3042 CCA 1683967.1 UUCAUGU AD-887347 A-1683968.1  823 CAUGAACGACUUCUUC 3025-3043 A-  824 GUGGAAGAAGUC 3025-3043 CAC 1683969.1 GUUCAUG AD-887348 A-1683970.1  825 UGAACGACUUCUUCCA 3027-3045 A-  826 GAGUGGAAGAAG 3027-3045 CUC 1683971.1 UCGUUCA AD-887349 A-1683972.1  827 CGACUUCUUCCACUCC 3031-3049 A-  828 GAAGGAGUGGAA 3031-3049 UUC 1683973.1 GAAGUCG AD-887350 A-1683974.1  829 UCCACUCCUUCCUGAU 3039-3057 A-  830 ACAAUCAGGAAG 3039-3057 UGU 1683975.1 GAGUGGA AD-887351 A-1683976.1  831 ACUCCUUCCUGAUUGU 3042-3060 A-  832 AACACAAUCAGGA 3042-3060 GUU 1683977.1 AGGAGU AD-887352 A-1683978.1  833 CUCCUUCCUGAUUGUG 3043-3061 A-  834 GAACACAAUCAGG 3043-3061 UUC 1683979.1 AAGGAG AD-887353 A-1683980.1  835 UCCUUCCUGAUUGUG 3044-3062 A-  836 GGAACACAAUCAG 3044-3062 UUCC 1683981.1 GAAGGA AD-887354 A-1683982.1  837 CUAUGUGCCUUAUUG 3123-3141 A-  838 UAAACAAUAAGG 3123-3141 UUUA 1683983.1 CACAUAG AD-887355 A-1683984.1  839 UGGUCCUAAACCUAUU 3171-3189 A-  840 AGAAAUAGGUUU 3171-3189 UCU 1683985.1 AGGACCA AD-887356 A-1683986.1  841 GGUCCUAAACCUAUUU 3172-3190 A-  842 CAGAAAUAGGUU 3172-3190 CUG 1683987.1 UAGGACC AD-887357 A-1683988.1  843 GUCCUAAACCUAUUUC 3173-3191 A-  844 CCAGAAAUAGGU 3173-3191 UGG 1683989.1 UUAGGAC AD-887358 A-1683990.1  845 CCUUACGUGAAUUUA 3312-3330 A-  846 AGAAUAAAUUCA 3312-3330 UUCU 1683991.1 CGUAAGG AD-887359 A-1683992.1  847 CAAAGGUCACAAUUUC 3439-3457 A-  848 GAGGAAAUUGUG 3439-3457 CUC 1683993.1 ACCUUUG AD-887360 A-1683994.1  849 UCACAAUUUCCUCAAG 3445-3463 A-  850 UUCCUUGAGGAA 3445-3463 GAA 1683995.1 AUUGUGA AD-887361 A-1683996.1  851 CCUCAAGGAAAAAGAU 3454-3472 A-  852 UUUAUCUUUUUC 3454-3472 AAA 1683997.1 CUUGAGG AD-887362 A-1683998.1  853 GCUUCAUUGUCCUCAU 3885-3903 A-  854 AUCAUGAGGACA 3885-3903 GAU 1683999.1 AUGAAGC AD-887363 A-1684000.1  855 CUUCAUUGUCCUCAUG 3886-3904 A-  856 GAUCAUGAGGAC 3886-3904 AUC 1684001.1 AAUGAAG AD-887364 A-1684002.1  857 UGCAGACAAGAUCUUC 3982-4000 A-  858 AGUGAAGAUCUU 3982-4000 ACU 1684003.1 GUCUGCA AD-887365 A-1684004.1  859 CAGACAAGAUCUUCAC 3984-4002 A-  860 UAAGUGAAGAUC 3984-4002 UUA 1684005.1 UUGUCUG AD-887366 A-1684006.1  861 AGACAAGAUCUUCACU 3985-4003 A-  862 GUAAGUGAAGAU 3985-4003 UAC 1684007.1 CUUGUCU AD-887367 A-1684008.1  863 GACAAGAUCUUCACUU 3986-4004 A-  864 UGUAAGUGAAGA 3986-4004 ACA 1684009.1 UCUUGUC AD-887368 A-1684010.1  865 ACAAGAUCUUCACUUA 3987-4005 A-  866 AUGUAAGUGAAG 3987-4005 CAU 1684011.1 AUCUUGU AD-887369 A-1684012.1  867 CAAGAUCUUCACUUAC 3988-4006 A-  868 GAUGUAAGUGAA 3988-4006 AUC 1684013.1 GAUCUUG AD-887370 A-1684014.1  869 AGAUCUUCACUUACAU 3990-4008 A-  870 AAGAUGUAAGUG 3990-4008 CUU 1684015.1 AAGAUCU AD-887371 A-1684016.1  871 GAUCUUCACUUACAUC 3991-4009 A-  872 GAAGAUGUAAGU 3991-4009 UUC 1684017.1 GAAGAUC AD-887372 A-1684018.1  873 UCUUCACUUACAUCUU 3993-4011 A-  874 AUGAAGAUGUAA 3993-4011 CAU 1684019.1 GUGAAGA AD-887373 A-1684020.1  875 CUUCACUUACAUCUUC 3994-4012 A-  876 AAUGAAGAUGUA 3994-4012 AUU 1684021.1 AGUGAAG AD-887374 A-1684022.1  877 UUCACUUACAUCUUCA 3995-4013 A-  878 GAAUGAAGAUGU 3995-4013 UUC 1684023.1 AAGUGAA AD-887375 A-1684024.1  879 UCACUUACAUCUUCAU 3996-4014 A-  880 AGAAUGAAGAUG 3996-4014 UCU 1684025.1 UAAGUGA AD-887376 A-1684026.1  881 CACUUACAUCUUCAUU 3997-4015 A-  882 CAGAAUGAAGAU 3997-4015 CUG 1684027.1 GUAAGUG AD-887377 A-1684028.1  883 CUUACAUCUUCAUUCU 3999-4017 A-  884 UCCAGAAUGAAG 3999-4017 GGA 1684029.1 AUGUAAG AD-887378 A-1684030.1  885 ACAUCUUCAUUCUGGA 4002-4020 A-  886 AUUUCCAGAAUG 4002-4020 AAU 1684031.1 AAGAUGU AD-887379 A-1684032.1  887 CAUCUUCAUUCUGGAA 4003-4021 A-  888 CAUUUCCAGAAU 4003-4021 AUG 1684033.1 GAAGAUG AD-887380 A-1684034.1  889 UCUUCAUUCUGGAAA 4005-4023 A-  890 AGCAUUUCCAGA 4005-4023 UGCU 1684035.1 AUGAAGA AD-887381 A-1684036.1  891 CUUCAUUCUGGAAAUG 4006-4024 A-  892 AAGCAUUUCCAG 4006-4024 CUU 1684037.1 AAUGAAG AD-887382 A-1684038.1  893 UCUGGAAAUGCUUCUA 4012-4030 A-  894 UUUUAGAAGCAU 4012-4030 AAA 1684039.1 UUCCAGA AD-887383 A-1684040.1  895 GCUGGAUUUCCUAAU 4078-4096 A-  896 AACAAUUAGGAA 4078-4096 UGUU 1684041.1 AUCCAGC AD-887384 A-1684042.1  897 CUGGAUUUCCUAAUU 4079-4097 A-  898 CAACAAUUAGGA 4079-4097 GUUG 1684043.1 AAUCCAG AD-887385 A-1684044.1  899 CCUCUAAGAGCCUUAU 4187-4205 A-  900 UAGAUAAGGCUC 4187-4205 CUA 1684045.1 UUAGAGG AD-887386 A-1684046.1  901 CUCUAAGAGCCUUAUC 4188-4206 A-  902 CUAGAUAAGGCU 4188-4206 UAG 1684047.1 CUUAGAG AD-887387 A-1684048.1  903 CUUCCAUCAUGAAUGU 4254-4272 A-  904 AGCACAUUCAUG 4254-4272 GCU 1684049.1 AUGGAAG AD-887388 A-1684050.1  905 UUUCCUGCAAGUCAAG 4373-4391 A-  906 GAACUUGACUUG 4373-4391 UUC 1684051.1 CAGGAAA AD-887389 A-1684052.1  907 CUGCAAGUCAAGUUCC 4377-4395 A-  908 UUUGGAACUUGA 4377-4395 AAA 1684053.1 CUUGCAG AD-887390 A-1684054.1  909 AGUCAAGUUCCAAAUC 4382-4400 A-  910 AACGAUUUGGAA 4382-4400 GUU 1684055.1 CUUGACU AD-887391 A-1684056.1  911 ACUUGGUUACCUAUCU 4477-4495 A-  912 CAGAGAUAGGUA 4477-4495 CUG 1684057.1 ACCAAGU AD-887392 A-1684058.1  913 CUUGGUUACCUAUCUC 4478-4496 A-  914 GCAGAGAUAGGU 4478-4496 UGC 1684059.1 AACCAAG AD-887393 A-1684060.1  915 GGUUACCUAUCUCUGC 4481-4499 A-  916 GAAGCAGAGAUA 4481-4499 UUC 1684061.1 GGUAACC AD-887394 A-1684062.1  917 GUUACCUAUCUCUGCU 4482-4500 A-  918 UGAAGCAGAGAU 4482-4500 UCA 1684063.1 AGGUAAC AD-887395 A-1684064.1  919 UUACCUAUCUCUGCUU 4483-4501 A-  920 UUGAAGCAGAGA 4483-4501 CAA 1684065.1 UAGGUAA AD-887396 A-1684066.1  921 UACCUAUCUCUGCUUC 4484-4502 A-  922 CUUGAAGCAGAG 4484-4502 AAG 1684067.1 AUAGGUA AD-887397 A-1684068.1  923 ACCUAUCUCUGCUUCA 4485-4503 A-  924 ACUUGAAGCAGA 4485-4503 AGU 1684069.1 GAUAGGU AD-887398 A-1684070.1  925 CCUAUCUCUGCUUCAA 4486-4504 A-  926 AACUUGAAGCAG 4486-4504 GUU 1684071.1 AGAUAGG AD-887399 A-1684072.1  927 CUAUCUCUGCUUCAAG 4487-4505 A-  928 CAACUUGAAGCA 4487-4505 UUG 1684073.1 GAGAUAG AD-887400 A-1684074.1  929 AUCUCUGCUUCAAGUU 4489-4507 A-  930 UGCAACUUGAAG 4489-4507 GCA 1684075.1 CAGAGAU AD-887401 A-1684076.1  931 UCUCUGCUUCAAGUU 4490-4508 A-  932 UUGCAACUUGAA 4490-4508 GCAA 1684077.1 GCAGAGA AD-887402 A-1684078.1  933 CUCUGCUUCAAGUUGC 4491-4509 A-  934 GUUGCAACUUGA 4491-4509 AAC 1684079.1 AGCAGAG AD-887403 A-1684080.1  935 UCUGCUUCAAGUUGCA 4492-4510 A-  936 AGUUGCAACUUG 4492-4510 ACU 1684081.1 AAGCAGA AD-887404 A-1684082.1  937 UAUCAUCUUUGGGUC 4618-4636 A-  938 GAAUGACCCAAAG 4618-4636 AUUC 1684083.1 AUGAUA AD-887405 A-1684084.1  939 AUCAUCUUUGGGUCA 4619-4637 A-  940 AGAAUGACCCAAA 4619-4637 UUCU 1684085.1 GAUGAU AD-887406 A-1684086.1  941 UCAUCUUUGGGUCAU 4620-4638 A-  942 AAGAAUGACCCAA 4620-4638 UCUU 1684087.1 AGAUGA AD-887407 A-1684088.1  943 CAUCUUUGGGUCAUU 4621-4639 A-  944 GAAGAAUGACCCA 4621-4639 CUUC 1684089.1 AAGAUG AD-887408 A-1684090.1  945 CUUUGGGUCAUUCUU 4624-4642 A-  946 AGUGAAGAAUGA 4624-4642 CACU 1684091.1 CCCAAAG AD-887409 A-1684092.1  947 UUGGGUCAUUCUUCA 4626-4644 A-  948 AAAGUGAAGAAU 4626-4644 CUUU 1684093.1 GACCCAA AD-887410 A-1684094.1  949 UGGGUCAUUCUUCAC 4627-4645 A-  950 CAAAGUGAAGAA 4627-4645 UUUG 1684095.1 UGACCCA AD-887411 A-1684096.1  951 GGGUCAUUCUUCACU 4628-4646 A-  952 UCAAAGUGAAGA 4628-4646 UUGA 1684097.1 AUGACCC AD-887412 A-1684098.1  953 GGUCAUUCUUCACUU 4629-4647 A-  954 UUCAAAGUGAAG 4629-4647 UGAA 1684099.1 AAUGACC AD-887413 A-1684100.1  955 GUCAUUCUUCACUUU 4630-4648 A-  956 GUUCAAAGUGAA 4630-4648 GAAC 1684101.1 GAAUGAC AD-887414 A-1684102.1  957 CAUUCUUCACUUUGAA 4632-4650 A-  958 AAGUUCAAAGUG 4632-4650 CUU 1684103.1 AAGAAUG AD-887415 A-1684104.1  959 UCACUUUGAACUUGU 4638-4656 A-  960 AUGAACAAGUUC 4638-4656 UCAU 1684105.1 AAAGUGA AD-887416 A-1684106.1  961 CUUGUUCAUUGGUGU 4648-4666 A-  962 GAUGACACCAAU 4648-4666 CAUC 1684107.1 GAACAAG AD-887417 A-1684108.1  963 GUGUCAUCAUAGAUAA 4659-4677 A-  964 AAAUUAUCUAUG 4659-4677 UUU 1684109.1 AUGACAC AD-887418 A-1684110.1  965 UGUCAUCAUAGAUAAU 4660-4678 A-  966 GAAAUUAUCUAU 4660-4678 UUC 1684111.1 GAUGACA AD-887419 A-1684112.1  967 GAGGUCAAGACAUCUU 4701-4719 A-  968 AUAAAGAUGUCU 4701-4719 UAU 1684113.1 UGACCUC AD-887420 A-1684114.1  969 AGGUCAAGACAUCUUU 4702-4720 A-  970 CAUAAAGAUGUC 4702-4720 AUG 1684115.1 UUGACCU AD-887421 A-1684116.1  971 GGUCAAGACAUCUUUA 4703-4721 A-  972 UCAUAAAGAUGU 4703-4721 UGA 1684117.1 CUUGACC AD-887422 A-1684118.1  973 CCACAAAAGCCAAUUC 4775-4793 A-  974 GAGGAAUUGGCU 4775-4793 CUC 1684119.1 UUUGUGG AD-887423 A-1684120.1  975 GACCUAGUGACAAAUC 4826-4844 A-  976 CUUGAUUUGUCA 4826-4844 AAG 1684121.1 CUAGGUC AD-887424 A-1684122.1  977 GUAUCAUGGUUCUUA 4857-4875 A-  978 CAGAUAAGAACCA 4857-4875 UCUG 1684123.1 UGAUAC AD-887425 A-1684124.1  979 UAUCAUGGUUCUUAU 4858-4876 A-  980 ACAGAUAAGAACC 4858-4876 CUGU 1684125.1 AUGAUA AD-887426 A-1684126.1  981 UCAUGGUUCUUAUCU 4860-4878 A-  982 AGACAGAUAAGA 4860-4878 GUCU 1684127.1 ACCAUGA AD-887427 A-1684128.1  983 CAUGGUUCUUAUCUG 4861-4879 A-  984 GAGACAGAUAAG 4861-4879 UCUC 1684129.1 AACCAUG AD-887428 A-1684130.1  985 AUGGUUCUUAUCUGU 4862-4880 A-  986 UGAGACAGAUAA 4862-4880 CUCA 1684131.1 GAACCAU AD-887429 A-1684132.1  987 UGGUUCUUAUCUGUC 4863-4881 A-  988 UUGAGACAGAUA 4863-4881 UCAA 1684133.1 AGAACCA AD-887430 A-1684134.1  989 GGUUCUUAUCUGUCU 4864-4882 A-  990 GUUGAGACAGAU 4864-4882 CAAC 1684135.1 AAGAACC AD-887431 A-1684136.1  991 GUUCUUAUCUGUCUC 4865-4883 A-  992 UGUUGAGACAGA 4865-4883 AACA 1684137.1 UAAGAAC AD-887432 A-1684138.1  993 UCUUAUCUGUCUCAAC 4867-4885 A-  994 CAUGUUGAGACA 4867-4885 AUG 1684139.1 GAUAAGA AD-887433 A-1684140.1  995 AUCUGUCUCAACAUGG 4871-4889 A-  996 UUACCAUGUUGA 4871-4889 UAA 1684141.1 GACAGAU AD-887434 A-1684142.1  997 UCUGUCUCAACAUGGU 4872-4890 A-  998 GUUACCAUGUUG 4872-4890 AAC 1684143.1 AGACAGA AD-887435 A-1684144.1  999 CUGUCUCAACAUGGUA 4873-4891 A- 1000 GGUUACCAUGUU 4873-4891 ACC 1684145.1 GAGACAG AD-887436 A-1684146.1 1001 UCCUGGUCAUGUUCA 5253-5271 A- 1002 UAGAUGAACAUG 5253-5271 UCUA 1684147.1 ACCAGGA AD-887437 A-1684148.1 1003 AGUUCAUCCUGGAAGU 5455-5473 A- 1004 UGAACUUCCAGG 5455-5473 UCA 1684149.1 AUGAACU AD-887438 A-1684150.1 1005 CCAUCUGUUGGAAUAU 5495-5513 A- 1006 AGAAUAUUCCAA 5495-5513 UCU 1684151.1 CAGAUGG AD-887439 A-1684152.1 1007 CAUCUGUUGGAAUAU 5496-5514 A- 1008 UAGAAUAUUCCA 5496-5514 UCUA 1684153.1 ACAGAUG AD-887440 A-1684154.1 1009 UCUGUUGGAAUAUUC 5498-5516 A- 1010 AGUAGAAUAUUC 5498-5516 UACU 1684155.1 CAACAGA AD-887441 A-1684156.1 1011 CAUACUGGAGAAUUU 5572-5590 A- 1012 ACUAAAAUUCUCC 5572-5590 UAGU 1684157.1 AGUAUG AD-887442 A-1684158.1 1013 CUCCUCUUCUCAUAGC 5730-5748 A- 1014 UUUGCUAUGAGA 5730-5748 AAA 1684159.1 AGAGGAG AD-887443 A-1684160.1 1015 UCCUCUUCUCAUAGCA 5731-5749 A- 1016 UUUUGCUAUGAG 5731-5749 AAA 1684161.1 AAGAGGA AD-887444 A-1684162.1 1017 CCUCUUCUCAUAGCAA 5732-5750 A- 1018 GUUUUGCUAUGA 5732-5750 AAC 1684163.1 GAAGAGG AD-887445 A-1684164.1 1019 CUCUUCUCAUAGCAAA 5733-5751 A- 1020 GGUUUUGCUAUG 5733-5751 ACC 1684165.1 AGAAGAG AD-887446 A-1684166.1 1021 GAUCCAUUGUCUUGAC 5803-5821 A- 1022 GAUGUCAAGACA 5803-5821 AUC 1684167.1 AUGGAUC AD-887447 A-1684168.1 1023 AUCCAUUGUCUUGACA 5804-5822 A- 1024 AGAUGUCAAGAC 5804-5822 UCU 1684169.1 AAUGGAU AD-887448 A-1684170.1 1025 UCCAUUGUCUUGACAU 5805-5823 A- 1026 AAGAUGUCAAGA 5805-5823 CUU 1684171.1 CAAUGGA AD-887449 A-1684172.1 1027 CAUUGUCUUGACAUCU 5807-5825 A- 1028 AUAAGAUGUCAA 5807-5825 UAU 1684173.1 GACAAUG AD-887450 A-1684174.1 1029 UUGUCUUGACAUCUU 5809-5827 A- 1030 AAAUAAGAUGUC 5809-5827 AUUU 1684175.1 AAGACAA AD-887451 A-1684176.1 1031 UGUCUUGACAUCUUA 5810-5828 A- 1032 CAAAUAAGAUGU 5810-5828 UUUG 1684177.1 CAAGACA AD-887452 A-1684178.1 1033 GUCUUGACAUCUUAU 5811-5829 A- 1034 GCAAAUAAGAUG 5811-5829 UUGC 1684179.1 UCAAGAC AD-887453 A-1684180.1 1035 GGAGAUGGAUUCUCU 5860-5878 A- 1036 ACGAAGAGAAUCC 5860-5878 UCGU 1684181.1 AUCUCC AD-887454 A-1684182.1 1037 GAGAUGGAUUCUCUU 5861-5879 A- 1038 AACGAAGAGAAU 5861-5879 CGUU 1684183.1 CCAUCUC AD-887455 A-1684184.1 1039 AGAUGGAUUCUCUUC 5862-5880 A- 1040 GAACGAAGAGAA 5862-5880 GUUC 1684185.1 UCCAUCU AD-887456 A-1684186.1 1041 GAUGGAUUCUCUUCG 5863-5881 A- 1042 UGAACGAAGAGA 5863-5881 UUCA 1684187.1 AUCCAUC AD-887457 A-1684188.1 1043 AUGGAUUCUCUUCGU 5864-5882 A- 1044 GUGAACGAAGAG 5864-5882 UCAC 1684189.1 AAUCCAU AD-887458 A-1684190.1 1045 UGGAUUCUCUUCGUU 5865-5883 A- 1046 UGUGAACGAAGA 5865-5883 CACA 1684191.1 GAAUCCA AD-887459 A-1684192.1 1047 GGAUUCUCUUCGUUC 5866-5884 A- 1048 CUGUGAACGAAG 5866-5884 ACAG 1684193.1 AGAAUCC AD-887460 A-1684194.1 1049 GAUUCUCUUCGUUCAC 5867-5885 A- 1050 UCUGUGAACGAA 5867-5885 AGA 1684195.1 GAGAAUC AD-887461 A-1684196.1 1051 UUCUCUUCGUUCACAG 5869-5887 A- 1052 CAUCUGUGAACG 5869-5887 AUG 1684197.1 AAGAGAA AD-887462 A-1684198.1 1053 UCUCUUCGUUCACAGA 5870-5888 A- 1054 CCAUCUGUGAAC 5870-5888 UGG 1684199.1 GAAGAGA AD-887463 A-1684200.1 1055 CUCUUCGUUCACAGAU 5871-5889 A- 1056 UCCAUCUGUGAA 5871-5889 GGA 1684201.1 CGAAGAG AD-887464 A-1684202.1 1057 UCUUCGUUCACAGAUG 5872-5890 A- 1058 UUCCAUCUGUGA 5872-5890 GAA 1684203.1 ACGAAGA AD-887465 A-1684204.1 1059 AGGUUCAUGUCUGCAA 5894-5912 A- 1060 GAUUUGCAGACA 5894-5912 AUC 1684205.1 UGAACCU AD-887466 A-1684206.1 1061 UCUGCAAAUCCUUCCA 5903-5921 A- 1062 CUUUGGAAGGAU 5903-5921 AAG 1684207.1 UUGCAGA AD-887467 A-1684208.1 1063 CUGCAAAUCCUUCCAA 5904-5922 A- 1064 ACUUUGGAAGGA 5904-5922 AGU 1684209.1 UUUGCAG AD-887468 A-1684210.1 1065 GUGUCUGCUACUGUC 5969-5987 A- 1066 GAAUGACAGUAG 5969-5987 AUUC 1684211.1 CAGACAC AD-887469 A-1684212.1 1067 UGUCUGCUACUGUCA 5970-5988 A- 1068 UGAAUGACAGUA 5970-5988 UUCA 1684213.1 GCAGACA AD-887470 A-1684214.1 1069 GUCUGCUACUGUCAU 5971-5989 A- 1070 CUGAAUGACAGU 5971-5989 UCAG 1684215.1 AGCAGAC AD-887471 A-1684216.1 1071 ACCGCUUAAGGCAAAA 6006-6024 A- 1072 ACAUUUUGCCUU 6006-6024 UGU 1684217.1 AAGCGGU AD-887472 A-1684218.1 1073 CCGCUUAAGGCAAAAU 6007-6025 A- 1074 GACAUUUUGCCU 6007-6025 GUC 1684219.1 UAAGCGG AD-887473 A-1684220.1 1075 UCUCCACCUUCAUAUG 6158-6176 A- 1076 UAUCAUAUGAAG 6158-6176 AUA 1684221.1 GUGGAGA AD-887474 A-1684222.1 1077 UGCCAAAAUCCUUUUU 6344-6362 A- 1078 GAUAAAAAGGAU 6344-6362 AUC 1684223.1 UUUGGCA AD-887475 A-1684224.1 1079 GCCAAAAUCCUUUUUA 6345-6363 A- 1080 UGAUAAAAAGGA 6345-6363 UCA 1684225.1 UUUUGGC AD-887476 A-1684226.1 1081 UCGUAAGAGAACUCUG 6463-6481 A- 1082 CUACAGAGUUCU 6463-6481 UAG 1684227.1 CUUACGA AD-887477 A-1684228.1 1083 UCUGCCUUGUCAUCUU 6563-6581 A- 1084 GAAAAGAUGACA 6563-6581 UUC 1684229.1 AGGCAGA AD-887478 A-1684230.1 1087 CUGCCUUGUCAUCUUU 6564-6582 A- 1086 UGAAAAGAUGAC 6564-6582 UCA 1684231.1 AAGGCAG AD-887479 A-1684232.1 1085 UGCCUUGUCAUCUUU 6565-6583 A- 1088 GUGAAAAGAUGA 6565-6583 UCAC 1684233.1 CAAGGCA AD-887480 A-1684234.1 1089 GCCUUGUCAUCUUUUC 6566-6584 A- 1090 UGUGAAAAGAUG 6566-6584 ACA 1684235.1 ACAAGGC AD-887481 A-1684236.1 1091 CCUUGUCAUCUUUUCA 6567-6585 A- 1092 CUGUGAAAAGAU 6567-6585 CAG 1684237.1 GACAAGG AD-887482 A-1684238.1 1093 CAUCUUUUCACAGGAU 6573-6591 A- 1094 ACAAUCCUGUGA 6573-6591 UGU 1684239.1 AAAGAUG AD-887483 A-1684240.1 1095 CCCAUGUAAAUAAACA 6606-6624 A- 1096 UGUUGUUUAUU 6606-6624 ACA 1684241.1 UACAUGGG AD-887484 A-1684242.1 1097 CAUUCAUCUUGACUCA 6911-6929 A- 1098 AUGUGAGUCAAG 6911-6929 CAU 1684243.1 AUGAAUG AD-887485 A-1684244.1 1099 ACAUAUUACACUCCUC 7040-7058 A- 1100 UUUGAGGAGUGU 7040-7058 AAA 1684245.1 AAUAUGU AD-887486 A-1684246.1 1101 CAUAUUACACUCCUCA 7041-7059 A- 1102 UUUUGAGGAGUG 7041-7059 AAA 1684247.1 UAAUAUG AD-887487 A-1684248.1 1103 UGCCCAAAAUACUGAU 7140-7158 A- 1104 AUUAUCAGUAUU 7140-7158 AAU 1684249.1 UUGGGCA AD-887488 A-1684250.1 1105 GCCCAAAAUACUGAUA 7141-7159 A- 1106 UAUUAUCAGUAU 7141-7159 AUA 1684251.1 UUUGGGC AD-887489 A-1684252.1 1107 CUGAUAAUAGUCUCU 7151-7169 A- 1108 UUUAAGAGACUA 7151-7169 UAAA 1684253.1 UUAUCAG AD-887490 A-1684254.1 1109 GUCAAAUUUUCCUGCU 7177-7195 A- 1110 GAAAGCAGGAAA 7177-7195 UUC 1684255.1 AUUUGAC AD-887491 A-1684256.1 1111 UCAAAUUUUCCUGCUU 7178-7196 A- 1112 AGAAAGCAGGAA 7178-7196 UCU 1684257.1 AAUUUGA AD-887492 A-1684258.1 1113 CAAAUUUUCCUGCUUU 7179-7197 A- 1114 AAGAAAGCAGGA 7179-7197 CUU 1684259.1 AAAUUUG AD-887493 A-1684260.1 1115 AUUGUUUAGUCAUCC 7205-7223 A- 1116 GAAAGGAUGACU 7205-7223 UUUC 1684261.1 AAACAAU AD-887494 A-1684262.1 1117 GCAUCACUUGUAUACA 7322-7340 A- 1118 GAUUGUAUACAA 7322-7340 AUC 1684263.1 GUGAUGC AD-887495 A-1684264.1 1119 CACCAACUUACUUUCC 7453-7471 A- 1120 UUAGGAAAGUAA 7453-7471 UAA 1684265.1 GUUGGUG AD-887496 A-1684266.1 1121 ACCAACUUACUUUCCU 7454-7472 A- 1122 UUUAGGAAAGUA 7454-7472 AAA 1684267.1 AGUUGGU AD-887497 A-1684268.1 1123 CCAACUUACUUUCCUA 7455-7473 A- 1124 AUUUAGGAAAGU 7455-7473 AAU 1684269.1 AAGUUGG AD-887498 A-1684270.1 1125 CAACUUACUUUCCUAA 7456-7474 A- 1126 AAUUUAGGAAAG 7456-7474 AUU 1684271.1 UAAGUUG AD-887499 A-1684272.1 1127 AGGAAGAUGUCACCUU 7517-7535 A- 5814 GAGAAGGUGACA 7517-7535 CUC 1684273.1 UCUUCCU AD-887500 A-1684274.1 1128 GAAGAUGUCACCUUCU 7519-7537 A- 1130 AGGAGAAGGUGA 7519-7537 CCU 1684275.1 CAUCUUC AD-887501 A-1684276.1 1131 AGAUGUCACCUUCUCC 7521-7539 A- 1132 UAAGGAGAAGGU 7521-7539 UUA 1684277.1 GACAUCU AD-887502 A-1684278.1 1133 GAUGUCACCUUCUCCU 7522-7540 A- 1134 UUAAGGAGAAGG 7522-7540 UAA 1684279.1 UGACAUC AD-887503 A-1684280.1 1135 AUGUCACCUUCUCCUU 7523-7541 A- 1136 UUUAAGGAGAAG 7523-7541 AAA 1684281.1 GUGACAU AD-887504 A-1684282.1 1137 UGUCACCUUCUCCUUA 7524-7542 A- 1138 UUUUAAGGAGAA 7524-7542 AAA 1684283.1 GGUGACA AD-887505 A-1684284.1 1139 GUCACCUUCUCCUUAA 7525-7543 A- 1140 AUUUUAAGGAGA 7525-7543 AAU 1684285.1 AGGUGAC AD-887506 A-1684286.1 1141 UCACCUUCUCCUUAAA 7526-7544 A- 1142 AAUUUUAAGGAG 7526-7544 AUU 1684287.1 AAGGUGA AD-887507 A-1684288.1 1143 ACCUUCUCCUUAAAAU 7528-7546 A- 1144 AGAAUUUUAAGG 7528-7546 UCU 1684289.1 AGAAGGU AD-887508 A-1684290.1 1145 CCUUCUCCUUAAAAUU 7529-7547 A- 1146 UAGAAUUUUAAG 7529-7547 CUA 1684291.1 GAGAAGG AD-887509 A-1684292.1 1147 CUUCUCCUUAAAAUUC 7530-7548 A- 1148 AUAGAAUUUUAA 7530-7548 UAU 1684293.1 GGAGAAG AD-887510 A-1684294.1 1149 UGAGAUCUUUCUUCU 7721-7739 A- 1150 UUAUAGAAGAAA 7721-7739 AUAA 1684295.1 GAUCUCA AD-887511 A-1684296.1 1151 GAUCUUUCUUCUAUA 7724-7742 A- 1152 ACUUUAUAGAAG 7724-7742 AAGU 1684297.1 AAAGAUC AD-887512 A-1684298.1 1153 UACCAUCUUAGGUUCA 8105-8123 A- 1154 GAAUGAACCUAA 8105-8123 UUC 1684299.1 GAUGGUA AD-887513 A-1684300.1 1155 ACCAUCUUAGGUUCAU 8106-8124 A- 1156 UGAAUGAACCUA 8106-8124 UCA 1684301.1 AGAUGGU AD-887514 A-1684302.1 1157 CCAUCUUAGGUUCAUU 8107-8125 A- 1158 AUGAAUGAACCU 8107-8125 CAU 1684303.1 AAGAUGG AD-887515 A-1684304.1 1159 CAUCUUAGGUUCAUUC 8108-8126 A- 1160 GAUGAAUGAACC 8108-8126 AUC 1684305.1 UAAGAUG AD-887516 A-1684306.1 1161 UCUUAGGUUCAUUCA 8110-8128 A- 1162 AAGAUGAAUGAA 8110-8128 UCUU 1684307.1 CCUAAGA AD-887517 A-1684308.1 1163 CUUAGGUUCAUUCAUC 8111-8129 A- 1164 UAAGAUGAAUGA 8111-8129 UUA 1684309.1 ACCUAAG AD-887518 A-1684310.1 1165 UUAGGUUCAUUCAUC 8112-8130 A- 1166 CUAAGAUGAAUG 8112-8130 UUAG 1684311.1 AACCUAA AD-887519 A-1684312.1 1167 UAGGUUCAUUCAUCU 8113-8131 A- 1168 CCUAAGAUGAAU 8113-8131 UAGG 1684313.1 GAACCUA AD-887520 A-1684314.1 1169 CUGCAUUAUGAAUACU 8368-8386 A- 1170 GUAAGUAUUCAU 8368-8386 UAC 1684315.1 AAUGCAG AD-887521 A-1684316.1 1171 ACACAAUUUCUUCUUA 8500-8518 A- 1172 UGCUAAGAAGAA 8500-8518 GCA 1684317.1 AUUGUGU AD-887522 A-1684318.1 1173 GUUCUUUUUCCUAUU 8541-8559 A- 1174 AUGAAAUAGGAA 8541-8559 UCAU 1684319.1 AAAGAAC AD-887523 A-1684320.1 1175 UCCUAUUUCAUGAACU 8549-8567 A- 1176 CAUAGUUCAUGA 8549-8567 AUG 1684321.1 AAUAGGA AD-887524 A-1684322.1 1177 CCUAUUUCAUGAACUA 8550-8568 A- 1178 ACAUAGUUCAUG 8550-8568 UGU 1684323.1 AAAUAGG AD-887525 A-1684324.1 1179 AUGUCUACUUGUGAC 8623-8641 A- 1180 AAAAGUCACAAG 8623-8641 UUUU 1684325.1 UAGACAU AD-887526 A-1684326.1 1181 UGUCUACUUGUGACU 8624-8642 A- 1182 AAAAAGUCACAAG 8624-8642 UUUU 1684327.1 UAGACA AD-887527 A-1684328.1 1183 UCUACUUGUGACUUU 8626-8644 A- 1184 AUAAAAAGUCAC 8626-8644 UUAU 1684329.1 AAGUAGA AD-887528 A-1684330.1 1185 CUACUUGUGACUUUU 8627-8645 A- 5815 GAUAAAAAGUCA 8627-8645 UAUC 1684331.1 CAAGUAG AD-887529 A-1684332.1 1186 GUUCUAAAUAGCUAU 9384-9402 A- 1188 UGAAAUAGCUAU 9384-9402 UUCA 1684333.1 UUAGAAC AD-887530 A-1684334.1 1189 GCUGUUUACAUAGGA 9600-9618 A- 1190 AGAAUCCUAUGU 9600-9618 UUCU 1684335.1 AAACAGC AD-887531 A-1684336.1 1191 GCUCAAAAUGUUUGA 9644-9662 A- 1192 AAACUCAAACAUU 9644-9662 GUUU 1684337.1 UUGAGC

TABLE 4A Exemplary Human SCN9A siRNA Modified Single Strands and Duplex Sequences Column 1 indicates duplex name and the number following the decimal point in a duplex name merely refers to a batch production number. Column 2 indicates the name of the sense sequence. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the modified sequence of a sense strand suitable for use in a duplex described herein. Column 5 indicates the antisense sequence name. Column 6 indicates the sequence ID for the sequence of column 7. Column 7 provides the sequence of a modified antisense strand suitable for use in a duplex described herein, e.g., a duplex comprising the sense sequence in the same row of the table. Column 8 indicates the position in the target mRNA (NM_001365536.1) that is complementary to the antisense strand of Column 7. Column 9 indicated the sequence ID for the sequence of column 8. Seq ID Sense Seq ID Antisense NO: mRNA target  Duplex sequence NO: Sense sequence sequence (anti Antisense sequence sequence in Seq ID NO: Name name (sense) (5′-3′) name sense) (5′-3′) NM_001365536.1 (mRNA target) AD- A- 1795 ususugu(Ahd)GfaUf A- 1796 VPusGfsuaaUfuGf CUUUUGUAGAUCUUG 3339 796825.1 1525636.1 CfUfugcaauuacaL96 1257916.1 CfaagaUfcUfacaa CAAUUACC asasg AD- A- 1797 ususcug(Uhd)GfuAf A- 1798 VPusGfsugaAfuUf GCUUCUGUGUAGGAG 3340 795366.1 1522818.1 GfGfagaauucacaL96 1522819.1 CfuccuAfcAfcaga AAUUCACU asgsc AD- A- 1799 asusgug(Ahd)AfaCf A- 1800 VPusAfscguAfaGf UUAUGUGAAACAAACC 3341 797565.2 1527044.1 AfAfaccuuacguaL96 1527045.1 GfuuugUfuUfcac UUACGUG ausasa AD- A- 1801 usgsuag(Ghd)AfgAf A- 1802 VPusGfsaaaAfgUf UGUGUAGGAGAAUUC 3342 795371.1 1522828.1 AfUfucacuuuucaL96 1522829.1 GfaauuCfuCfcuac ACUUUUCU ascsa AD- A- 1803 usasugu(Ghd)AfaAf A- 1804 VPusCfsguaAfgGf AUUAUGUGAAACAAAC 3343 797564.2 1527042.1 CfAfaaccuuacgaL96 1527043.1 UfuuguUfuCfaca CUUACGU uasasu AD- A- 1805 asgscau(Ahd)AfaUf A- 1806 VPusAfsuuuCfgAf GAAGCAUAAAUGUUU 3344 795634.2 1523299.1 GfUfuuucgaaauaL96 1523300.1 AfaacaUfuUfaugc UCGAAAUU USUSC AD- A- 1807 gsasucu(Uhd)CfuUf A- 1808 VPusUfscacUfaCf AUGAUCUUCUUUGUC 3345 795913.1 1523849.1 UfGfucguagugaaL96 1523850.1 GfacaaAfgAfagau GUAGUGAU csasu AD- A- 1809 gsgscgu(Uhd)GfuAf A- 1810 VPusGfsagaUfaGf AAGGCGUUGUAGUUC 3346 796618.1 1525247.1 GfUfuccuaucucaL96 1525248.1 GfaacuAfcAfacgc CUAUCUCC csusu AD- A- 1811 asuscuu(Chd)UfuUf A- 1812 VPusAfsucaCfuAf UGAUCUUCUUUGUCG 3347 795914.1 1523851.1 GfUfcguagugauaL96 1523852.1 CfgacaAfaGfaaga UAGUGAUU uscsa AD- A- 1813 usgsguu(Uhd)CfaGf A- 1814 VPusCfsugaAfuCf UGUGGUUUCAGCACA 3348 795739.1 1523509.1 CfAfcagauucagaL96 1523510.1 UfgugcUfgAfaacc GAUUCAGG ascsa AD- A- 1815 usgsucg(Ahd)GfuAf A- 1816 VPusCfsaguAfaAf AAUGUCGAGUACACUU 3349 795305.1 1522697.1 CfAfcuuuuacugaL96 1522698.1 AfguguAfcUfcgac UUACUGG asusu AD- A- 1817 asasgca(Ghd)AfaGf A- 1818 VPusAfsguaUfuCf ACAAGCAGAAGAUCUG 3350 797636.2 1527186.1 AfUfcugaauacuaL96 1527187.1 AfgaucUfuCfugcu AAUACUA usgsu AD- A- 1819 csasagu(Ghd)UfuCf A- 1820 VPusCfsaugAfcAf UUCAAGUGUUCCUACU 3351 802471.2 1536717.1 CfUfacugucaugaL96 1536718.1 GfuaggAfaCfacuu GUCAUGA gsasa AD- A- 1821 asusgcu(Ghd)AfgAf A- 1822 VPusUfsuucGfaCf AGAUGCUGAGAAAUU 3352 796209.1 1524439.1 AfAfuugucgaaaaL96 1524440.1 AfauuuCfuCfagca GUCGAAAU uscsu AD- A- 1823 asusguu(Uhd)CfuAf A- 1824 VPusAfsucaAfaUf GUAUGUUUCUAGCUG 3353 799223.1 1530270.1 GfCfugauuugauaL96 1530271.1 CfagcuAfgAfaaca AUUUGAUU usasc AD- A- 1825 gsasgau(Ghd)GfaUf A- 1826 VPusGfsaacGfaAf GGGAGAUGGAUUCUC 3354 799938.1 1531655.1 UfCfucuucguucaL96 1531656.1 GfagaaUfcCfaucu UUCGUUCA cscsc AD- A- 1827 ususgug(Ahd)CfuUf A- 1828 VPusCfsacuAfaAf UAUUGUGACUUUAAG 3355 797036.1 1526036.1 UfAfaguuuagugaL96 1526037.1 CfuuaaAfgUfcaca UUUAGUGG asusa AD- A- 1829 asusgau(Chd)UfuCf A- 1830 VPusAfscuaCfgAf ACAUGAUCUUCUUUG 3356 795911.1 1523845.1 UfUfugucguaguaL96 1523846.1 CfaaagAfaGfauca UCGUAGUG usgsu AD- A- 1831 asasggg(Ahd)AfaAf A- 1832 VPusAfscggAfaGf CAAAGGGAAAACAAUC 3357 795132.1 1522351.1 CfAfaucuuccguaL96 1522352.1 AfuuguUfuUfcccu UUCCGUU ususg AD- A- 1833 csusucu(Ghd)AfaAf A- 1834 VPusCfsaguUfuGf UUCUUCUGAAACAUCC 3358 796138.1 1524297.1 CfAfuccaaacugaL96 1524298.1 GfauguUfuCfagaa AAACUGA gsasa AD- A- 1835 ususgcu(Ahd)UfaGf A- 1836 VPusGfsaccAfaAf ACUUGCUAUAGGAAAU 3359 796919.1 1525802.1 GfAfaauuuggucaL96 1525803.1 UfuuccUfaUfagca UUGGUCU asgsu AD- A- 1837 usasuug(Uhd)GfaCf A- 1838 VPusCfsuaaAfcUf CUUAUUGUGACUUUA 3360 797034.1 1526032.1 UfUfuaaguuuagaL96 1526033.1 UfaaagUfcAfcaau AGUUUAGU asasg AD- A- 1839 ususggc(Ahd)GfaAf A- 1840 VPusAfsuaaUfcAf AAUUGGCAGAAACCCU 3361 795774.1 1523579.1 AfCfccugauuauaL96 1523580.1 GfgguuUfcUfgcca GAUUAUG asusu AD- A- 1841 ascsaug(Ahd)UfcUf A- 1842 VPusUfsacgAfcAf CUACAUGAUCUUCUUU 3362 795909.1 1523841.1 UfCfuuugucguaaL96 1523842.1 AfagaaGfaUfcaug GUCGUAG usasg AD- A- 1843 asgscuu(Ghd)AfaGf A- 1844 VPusGfsucuAfaUf UAAGCUUGAAGUAAAA 3363 802123.1 1536023.1 UfAfaaauuagacaL96 1536024.1 UfuuacUfuCfaagc UUAGACC ususa AD- A- 1845 uscscaa(Ahd)UfcGf A- 1846 VPusAfsacaUfuCf GUUCCAAAUCGUUCCG 3364 798588.2 1529045.1 UfUfccgaauguuaL96 1529046.1 GfgaacGfaUfuugg AAUGUUU asasc AD- A- 1847 asuscug(Ahd)GfaCf A- 1848 VPusCfsggcAfaAf GGAUCUGAGACUGAA 3365 796396.1 1524811.1 UfGfaauuugccgaL96 1524812.1 UfucagUfcUfcaga UUUGCCGA uscsc AD- A- 1849 gscsguu(Ghd)UfaGf A- 1850 VPusGfsgagAfuAf AGGCGUUGUAGUUCC 3366 796619.1 1525249.1 UfUfccuaucuccaL96 1525250.1 GfgaacUfaCfaacg UAUCUCCU CSCSU AD- A- 1851 usasuau(Uhd)UfuAf A- 1852 VPusAfsacgGfaUf GAUAUAUUUUACAACA 3367 801647.1 1535071.1 CfAfacauccguuaL96 1535072.1 GfuuguAfaAfaua UCCGUUA uasusc AD- A- 1853 asusguc(Ghd)AfgUf A- 1854 VPusAfsguaAfaAf AAAUGUCGAGUACACU 3368 795304.1 1522695.1 AfCfacuuuuacuaL96 1522696.1 GfuguaCfuCfgaca UUUACUG ususu AD- A- 1855 usgsaua(Ghd)UfuAf A- 1856 VPusUfsgcaAfaCf UUUGAUAGUUACCUA 3369 802553.1 1536879.1 CfCfuaguuugcaaL96 1536880.1 UfagguAfaCfuauc GUUUGCAA asasa AD- A- 1857 gsascuu(Ahd)CfcUf A- 1858 VPusCfsaauAfcUf AAGACUUACCUUUAGA 3370 800819.1 1533415.1 UfUfagaguauugaL96 1533416.1 CfuaaaGfgUfaagu GUAUUGU csusu AD- A- 1859 csusaaa(Uhd)UfaUf A- 1860 VPusAfsgauUfaCf UCCUAAAUUAUGGAAG 3371 801263.1 1534303.1 GfGfaaguaaucuaL96 1534304.1 UfuccaUfaAfuuua UAAUCUU gsgsa AD- A- 1861 asgsuca(Ahd)GfuUf A- 1862 VPusGfsaacGfaUf CAAGUCAAGUUCCAAA 3372 798580.1 1529029.1 CfCfaaaucguucaL96 1529030.1 UfuggaAfcUfugac UCGUUCC ususg AD- A- 1863 usgsauc(Uhd)UfcUf A- 1864 VPusCfsacuAfcGf CAUGAUCUUCUUUGU 3373 795912.1 1523847.1 UfUfgucguagugaL96 1523848.1 AfcaaaGfaAfgauc CGUAGUGA asusg AD- A- 1865 gsusuug(Ahd)AfcAf A- 1866 VPusCfsgaaAfgAf AGGUUUGAACACAAAU 3374 802503.1 1536779.1 CfAfaaucuuucgaL96 1536780.1 UfuuguGfuUfcaa CUUUCGG acscsu AD- A- 1867 asasguu(Chd)CfaAf A- 1868 VPusUfsucgGfaAf UCAAGUUCCAAAUCGU 3375 798584.2 1529037.1 AfUfcguuccgaaaL96 1529038.1 CfgauuUfgGfaacu UCCGAAU usgsa AD- A- 1869 usgsuag(Ahd)UfcUf A- 1870 VPusUfsgguAfaUf UUUGUAGAUCUUGCA 3376 796827.1 1525638.1 UfGfcaauuaccaaL96 1257918.1 UfgcaaGfaUfcuac AUUACCAU asasa AD- A- 1871 csasuga(Uhd)CfuUf A- 1872 VPusCfsuacGfaCf UACAUGAUCUUCUUU 3377 795910.1 1523843.1 CfUfuugucguagaL96 1523844.1 AfaagaAfgAfucau GUCGUAGU gsusa AD- A- 1873 ususgau(Ahd)GfuUf A- 1874 VPusGfscaaAfcUf UUUUGAUAGUUACCU 3378 802552.1 1536877.1 AfCfcuaguuugcaL96 1536878.1 AfgguaAfcUfauca AGUUUGCA asasa AD- A- 1875 csasccu(Uhd)CfuCfC A- 1876 VPusAfsgaaUfuUf GUCACCUUCUCCUUAA 3379 801304.1 1534385.1 fUfuaaaauucuaL96 1534386.1 UfaaggAfgAfaggu AAUUCUA gsasc AD- A- 1877 csusgau(Uhd)UfcCf A- 1878 VPusCfsaccUfuUf CUCUGAUUUCCUAAGA 3380 800334.1 1532445.1 UfAfagaaaggugaL96 1532446.1 CfuuagGfaAfauca AAGGUGG gsasg AD- A- 1879 usgsaga(Chd)UfgAf A- 1880 VPusAfsuuaCfaAf CUUGAGACUGACACAU 3381 802946.1 1537662.1 CfAfcauuguaauaL96 1537663.1 UfguguCfaGfucuc UGUAAUA asasg AD- A- 1881 csusgaa(Uhd)AfuAf A- 1882 VPusCfscuaAfuAf GGCUGAAUAUACAAGU 3382 796087.1 1524195.1 CfAfaguauuaggaL96 1524196.1 CfuuguAfuAfuuca AUUAGGA gscsc AD- A- 1883 csasacc(Chd)AfaAfA A- 1884 VPusAfsugcUfaAf CACAACCCAAAAUACU 3383 802625.2 1537023.1 fUfacuuagcauaL96 1537024.1 GfuauuUfuGfggu UAGCAUG ugsusg AD- A- 1885 csusgau(Ahd)AfuAf A- 1886 VPusGfsuuuAfaGf UACUGAUAAUAGUCUC 3384 800966.1 1533709.1 GfUfcucuuaaacaL96 1533710.1 AfgacuAfuUfauca UUAAACU gsusa AD- A- 1887 ususugu(Chd)GfuAf A- 1888 VPusAfsggaAfaAf UCUUUGUCGUAGUGA 3385 795920.1 1523863.1 GfUfgauuuuccuaL96 1523864.1 UfcacuAfcGfacaa UUUUCCUG asgsa AD- A- 1889 usgsaau(Ahd)UfaCf A- 1890 VPusUfsccuAfaUf GCUGAAUAUACAAGUA 3386 796088.1 1524197.1 AfAfguauuaggaaL96 1524198.1 AfcuugUfaUfauuc UUAGGAG asgsc AD- A- 1891 asgsaug(Ghd)AfuUf A- 1892 VPusUfsgaaCfgAf GGAGAUGGAUUCUCU 3387 799939.1 1531657.1 CfUfcuucguucaaL96 1531658.1 AfgagaAfuCfcauc UCGUUCAC uscsc AD- A- 1893 asasuau(Chd)AfuAf A- 1894 VPusGfsuaaAfcAf UGAAUAUCAUAAAGCU 3388 802853.2 1537477.1 AfAfgcuguuuacaL96 1537478.1 GfcuuuAfuGfaua GUUUACA uuscsa AD- A- 1895 uscsuuu(Ahd)UfaCf A- 1896 VPusAfsaccUfaAf AUUCUUUAUACCAUCU 3389 801724.1 1535225.1 CfAfucuuagguuaL96 1535226.1 GfauggUfaUfaaag UAGGUUC asasu AD- A- 1897 gscsaaa(Ghd)GfuCf A- 1898 VPusGfsaggAfaAf GAGCAAAGGUCACAAU 3390 797699.1 1527312.1 AfCfaauuuccucaL96 1527313.1 UfugugAfcCfuuug UUCCUCA csusc AD- A- 1899 asgsuca(Chd)CfaCf A- 1900 VPusAfscgaAfuGf UCAGUCACCACUCAGC 3391 796304.1 1524627.1 UfCfagcauucguaL96 1524628.1 CfugagUfgGfugac AUUCGUG usgsa AD- A- 1901 usgscua(Uhd)AfgGf A- 1902 VPusAfsgacCfaAf CUUGCUAUAGGAAAU 3392 796920.1 1525804.1 AfAfauuuggucuaL96 1525805.1 AfuuucCfuAfuagc UUGGUCUU asasg AD- A- 1903 gsascag(Ahd)GfaUf A- 1904 VPusAfsguaAfaUf GAGACAGAGAUGAUGA 3393 800110.1 1531997.1 GfAfugauuuacuaL96 1531998.1 CfaucaUfcUfcugu UUUACUC csusc AD- A- 1905 asasguc(Ahd)AfgUf A- 1906 VPusAfsacgAfuUf GCAAGUCAAGUUCCAA 3394 798579.1 1529027.1 UfCfcaaaucguuaL96 1529028.1 UfggaaCfuUfgacu AUCGUUC usgsc AD- A- 1907 usasggc(Uhd)AfaUf A- 1908 VPusAfsaucUfuGf UUUAGGCUAAUGACCC 3395 795841.1 1523713.1 GfAfcccaagauuaL96 1523714.1 GfgucaUfuAfgccu AAGAUUA asasa AD- A- 1909 asasgag(Chd)UfuAf A- 1910 VPusCfsuuaUfaCf GAAAGAGCUUAUUAA 3396 802105.2 1535987.1 UfUfaaguauaagaL96 1535988.1 UfuaauAfaGfcucu GUAUAAGC USUSC AD- A- 1911 usgsgaa(Uhd)AfuUf A- 1912 VPusUfsaacAfaAf GUUGGAAUAUUCUAC 3397 799594.1 1531002.1 CfUfacuuuguuaaL96 1531003.1 GfuagaAfuAfuucc UUUGUUAG asasc AD- A- 1913 asusgua(Chd)AfgAf A- 1914 VPusAfsuagAfaUf CAAUGUACAGAGGUUA 3398 800661.1 1533099.1 GfGfuuauucuauaL9 1533100.1 AfaccuCfuGfuaca UUCUAUA 6 ususg AD- A- 1915 asuscgu(Ahd)AfgAf A- 1916 VPusCfsuacAfgAf GAAUCGUAAGAGAACU 3399 800400.1 1532577.1 GfAfacucuguagaL96 1532578.1 GfuucuCfuUfacga CUGUAGG USUSC AD- A- 1917 csasucu(Ghd)UfuGf A- 1918 VPusGfsuagAfaUf CCCAUCUGUUGGAAUA 3400 799587.1 1530988.1 GfAfauauucuacaL96 1530989.1 AfuuccAfaCfagau UUCUACU gsgsg AD- A- 1919 gsuscuu(Uhd)AfcUf A- 1920 VPusGfscaaAfgAf UGGUCUUUACUGGAA 3401 796936.1 1525836.1 GfGfaaucuuugcaL96 1525837.1 UfuccaGfuAfaaga UCUUUGCA cscsa AD- A- 1921 csasaca(Chd)AfaUf A- 1922 VPusGfscuaAfgAf AACAACACAAUUUCUU 3402 802014.1 1535805.1 UfUfcuucuuagcaL96 1535806.1 AfgaaaUfuGfugu CUUAGCA ugsusu AD- A- 1923 usgsgau(Uhd)CfuCf A- 1924 VPusCfsuguGfaAf GAUGGAUUCUCUUCG 3403 799942.1 1531663.1 UfUfcguucacagaL96 1531664.1 CfgaagAfgAfaucc UUCACAGA asusc AD- A- 1925 gsusaug(Uhd)UfuCf A- 1926 VPusCfsaaaUfcAf AGGUAUGUUUCUAGC 3404 799221.1 1530266.1 UfAfgcugauuugaL96 1530267.1 GfcuagAfaAfcaua UGAUUUGA cscsu AD- A- 1927 cscsuuc(Chd)UfgAf A- 1928 VPusCfsuaaCfuGf AUCCUUCCUGAUAUGC 3405 801062.1 1533901.1 UfAfugcaguuagaL96 1533902.1 CfauauCfaGfgaag AGUUAGU gsasu AD- A- 1929 gsgsaga(Uhd)GfgAf A- 1930 VPusAfsacgAfaGf GGGGAGAUGGAUUCU 3406 799937.1 1531653.1 UfUfcucuucguuaL96 1531654.1 AfgaauCfcAfucuc CUUCGUUC CSCSC AD- A- 1931 gsusaga(Ahd)AfaCf A- 1932 VPusCfsagaUfgUf AUGUAGAAAACUUUU 3407 800461.1 1532699.1 UfUfuuacaucugaL96 1532700.1 AfaaagUfuUfucua ACAUCUGC csasu AD- A- 1933 asgscgu(Ghd)CfuUf A- 1934 VPusGfsuaaCfgUf UCAGCGUGCUUAUAGA 3408 800058.1 1531895.1 AfUfagacguuacaL96 1531896.1 CfuauaAfgCfacgc CGUUACC usgsa AD- A- 1935 gsusuuc(Uhd)AfgCf A- 1936 VPusCfsaauCfaAf AUGUUUCUAGCUGAU 3409 799225.1 1530274.1 UfGfauuugauugaL9 1530275.1 AfucagCfuAfgaaa UUGAUUGA 6 csasu AD- A- 1937 gscscca(Ahd)AfaUfA A- 1938 VPusCfsuauUfaUf CUGCCCAAAAUACUGA 3410 800956.1 1533689.1 fCfugauaauagaL96 1533690.1 CfaguaUfuUfuggg UAAUAGU csasg AD- A- 1939 ususugu(Chd)CfuAf A- 1940 VPusUfsauaCfgUf CAUUUGUCCUAAUCUA 3411 801681.2 1535139.1 AfUfcuacguauaaL96 1535140.1 AfgauuAfgGfacaa CGUAUAA asusg AD- A- 1941 usasauc(Ghd)CfuGf A- 1942 VPusUfsguaAfuAf UAUAAUCGCUGAACUU 3412 802206.2 1536189.1 AfAfcuuauuacaaL96 1536190.1 AfguucAfgCfgauu AUUACAC asusa AD- A- 1943 ususuga(Ahd)UfuCf A- 1944 VPusAfsacgGfuAf AAUUUGAAUUCAAUC 3413 801883.2 1535543.1 AfAfucuaccguuaL96 1535544.1 GfauugAfaUfucaa UACCGUUA asusu AD- A- 1945 csuscuu(Uhd)UfgAf A- 1946 VPusCfsauaGfaCf AACUCUUUUGAGGAA 3414 800273.2 1532323.1 GfGfaagucuaugaL96 1532324.1 UfuccuCfaAfaaga GUCUAUGC gsusu AD- A- 1947 asgscug(Ahd)UfuUf A- 1948 VPusAfscguUfuCf CUAGCUGAUUUGAUU 3415 799231.2 1530286.1 GfAfuugaaacguaL96 1530287.1 AfaucaAfaUfcagc GAAACGUA usasg AD- A- 1949 csusuua(Uhd)AfcCf A- 1950 VPusGfsaacCfuAf UUCUUUAUACCAUCUU 3416 801725.1 1535227.1 AfUfcuuagguucaL96 1535228.1 AfgaugGfuAfuaaa AGGUUCA gsasa AD- A- 1951 ususgca(Ahd)GfcCf A- 1952 VPusCfsucaCfaUf GGUUGCAAGCCUCUUA 3417 794914.1 1521918.1 UfCfuuaugugagaL96 1521919.1 AfagagGfcUfugca UGUGAGG ascsc AD- A- 1953 ususauu(Ghd)CfaUf A- 1954 VPusGfsuauAfcAf AUUUAUUGCAUCACU 3418 801132.1 1534041.1 CfAfcuuguauacaL96 1534042.1 AfgugaUfgCfaaua UGUAUACA asasu AD- A- 1955 ususuca(Chd)AfgGf A- 1956 VPusCfsuaaUfuAf CUUUUCACAGGAUUG 3419 800492.2 1532761.1 AfUfuguaauuagaL96 1532762.1 CfaaucCfuGfugaa UAAUUAGU asasg AD- A- 1957 csusuuu(Chd)AfcAf A- 1958 VPusAfsauuAfcAf AUCUUUUCACAGGAU 3420 800490.1 1532757.1 GfGfauuguaauuaL96 1532758.1 AfuccuGfuGfaaaa UGUAAUUA gsasu AD- A- 1959 csusgua(Ghd)GfaAf A- 1960 VPusAfsuaaUfcAf CUCUGUAGGAAUUAU 3421 800414.2 1532605.1 UfUfauugauuauaL96 1532606.1 AfuaauUfcCfuaca UGAUUAUA gsasg AD- A- 1961 ususccu(Ghd)AfuAf A- 1962 VPusAfsacuAfaCf CCUUCCUGAUAUGCAG 3422 801064.1 1533905.1 UfGfcaguuaguuaL96 1533906.1 UfgcauAfuCfagga UUAGUUG asgsg AD- A- 1963 gscsaag(Uhd)CfaAf A- 1964 VPusCfsgauUfuGf CUGCAAGUCAAGUUCC 3423 798577.1 1529023.1 GfUfuccaaaucgaL96 1529024.1 GfaacuUfgAfcuug AAAUCGU csasg AD- A- 1965 gsgsaag(Ahd)AfaGf A- 1966 VPusCfsagaCfaUf AUGGAAGAAAGGUUC 3424 799959.1 1531697.1 GfUfucaugucugaL96 1531698.1 GfaaccUfuUfcuuc AUGUCUGC csasu AD- A- 1967 asuscua(Ghd)GfgCf A- 1968 VPusAfsagaAfuCf CAAUCUAGGGCUAAAG 3425 801708.2 1535193.1 UfAfaagauucuuaL96 1535194.1 UfuuagCfcCfuaga AUUCUUU ususg AD- A- 1969 usasgcu(Ghd)AfuUf A- 1970 VPusCfsguuUfcAf UCUAGCUGAUUUGAU 3426 799230.2 1530284.1 UfGfauugaaacgaL96 1530285.1 AfucaaAfuCfagcu UGAAACGU asgsa AD- A- 1971 csusucc(Uhd)GfaUf A- 1972 VPusAfscuaAfcUf UCCUUCCUGAUAUGCA 3427 801063.1 1533903.1 AfUfgcaguuaguaL96 1533904.1 GfcauaUfcAfggaa GUUAGUU gsgsa AD- A- 1973 ascsuga(Uhd)GfaUf A- 1974 VPusAfsuucUfuAf GCACUGAUGAUUCUU 3428 800382.2 1532541.1 UfCfuuuaagaauaL96 1532542.1 AfagaaUfcAfucag UAAGAAUC usgsc AD- A- 1975 asgsacg(Uhd)UfaCf A- 1976 VPusUfsgccUfuAf AUAGACGUUACCGCUU 3429 800069.1 1531917.1 CfGfcuuaaggcaaL96 1531918.1 AfgcggUfaAfcguc AAGGCAA usasu AD- A- 1977 uscsgug(Ghd)CfuCf A- 1978 VPusCfsagaAfaAf AUUCGUGGCUCCUUG 3430 796318.1 1524655.1 CfUfuguuuucugaL96 1524656.1 CfaaggAfgCfcacg UUUUCUGC asasu AD- A- 1979 cscsuuu(Chd)UfuCf A- 1980 VPusGfsggaUfaUf AGCCUUUCUUCUUUCA 3431 800849.2 1533475.1 UfUfucauaucccaL96 1533476.1 GfaaagAfaGfaaag UAUCCCU gscsu AD- A- 1981 csasucu(Uhd)UfuCf A- 1982 VPusUfsacaAfuCf GUCAUCUUUUCACAGG 3432 800487.1 1532751.1 AfCfaggauuguaaL96 1532752.1 CfugugAfaAfagau AUUGUAA gsasc AD- A- 1983 csusguu(Ghd)GfaAf A- 1984 VPusUfscaaAfaCf GCCUGUUGGAAAUAG 3433 801835.1 1535447.1 AfUfagguuuugaaL96 1535448.1 CfuauuUfcCfaaca GUUUUGAU gsgsc AD- A- 1985 gsgsgag(Ahd)UfgGf A- 1986 VPusAfscgaAfgAf UGGGGAGAUGGAUUC 3434 799936.1 1531651.1 AfUfucucuucguaL96 1531652.1 GfaaucCfaUfcucc UCUUCGUU cscsa AD- A- 1987 ususgaa(Uhd)UfcAf A- 1988 VPusUfsaacGfgUf AUUUGAAUUCAAUCU 3435 801884.2 1535545.1 AfUfcuaccguuaaL96 1535546.1 AfgauuGfaAfuuca ACCGUUAU asasu AD- A- 1989 uscsauc(Uhd)UfaGf A- 1990 VPusGfsuucAfaAf AUUCAUCUUAGGCUA 3436 801747.2 1535271.1 GfCfuauuugaacaL96 1535272.1 UfagccUfaAfgaug UUUGAACC asasu AD- A- 1991 usgsauu(Chd)UfuUf A- 1992 VPusUfsuacGfaUf GAUGAUUCUUUAAGA 3437 800387.2 1532551.1 AfAfgaaucguaaaL96 1532552.1 UfcuuaAfaGfaauc AUCGUAAG asusc AD- A- 1993 gsusaau(Ghd)GfaCf A- 1994 VPusUfscauAfaCf AAGUAAUGGACAUUA 3438 800606.2 1532989.1 AfUfuaguuaugaaL96 1532990.1 UfaaugUfcCfauua GUUAUGAA csusu AD- A- 1995 ususgag(Ahd)CfuGf A- 1996 VPusUfsuacAfaUf ACUUGAGACUGACACA 3439 802945.2 1537660.1 AfCfacauuguaaaL96 1537661.1 GfugucAfgUfcuca UUGUAAU asgsu AD- A- 1997 gsasauu(Chd)AfaUf A- 1998 VPusAfsauaAfcGf UUGAAUUCAAUCUACC 3440 801886.2 1535549.1 CfUfaccguuauuaL96 1535550.1 GfuagaUfuGfaau GUUAUUU ucsasa AD- A- 1999 asusgau(Uhd)CfuUf A- 2000 VPusUfsacgAfuUf UGAUGAUUCUUUAAG 3441 800386.2 1532549.1 UfAfagaaucguaaL96 1532550.1 CfuuaaAfgAfauca AAUCGUAA uscsa AD- A- 2001 asgsccu(Ghd)UfuGf A- 2002 VPusAfsaacCfuAf CAAGCCUGUUGGAAAU 3442 801832.1 1535441.1 GfAfaauagguuuaL96 1535442.1 UfuuccAfaCfaggc AGGUUUU ususg AD- A- 2003 csgsugc(Uhd)UfaUf A- 2004 VPusCfsgguAfaCf AGCGUGCUUAUAGACG 3443 800060.1 1531899.1 AfGfacguuaccgaL96 1531900.1 GfucuaUfaAfgcac UUACCGC gscsu AD- A- 2005 ususuag(Uhd)GfgCf A- 2006 VPusCfsaagAfgUf ACUUUAGUGGCAAACA 3444 798332.1 1528540.1 AfAfacacucuugaL96 1528541.1 GfuuugCfcAfcuaa CUCUUGG asgsu AD- A- 2007 ascscuc(Uhd)CfuUf A- 2008 VPusAfsucuAfcAf AGACCUCUCUUUCCAU 3445 802141.2 1536059.1 UfCfcauguagauaL96 1536060.1 UfggaaAfgAfgagg GUAGAUU USCSU AD- A- 2009 csasacu(Uhd)AfcUf A- 2010 VPusUfsaauUfuAf ACCAACUUACUUUCCU 3446 801251.1 1534279.1 UfUfccuaaauuaaL96 1534280.1 GfgaaaGfuAfaguu AAAUUAU gsgsu AD- A- 2011 gscsuga(Ahd)CfcUf A- 2012 VPusUfscggAfaUf AGGCUGAACCUAUGAA 3447 797963.1 1527829.1 AfUfgaauuccgaaL96 1527830.1 UfcauaGfgUfucag UUCCGAU CSCSU AD- A- 2013 usasuca(Ahd)AfaUf A- 2014 VPusCfscuuCfgAf UUUAUCAAAAUAUUC 3448 800297.2 1532371.1 AfUfucucgaaggaL96 1532372.1 GfaauaUfuUfuga UCGAAGGC uasasa AD- A- 2015 ascsauc(Chd)GfuUf A- 2016 VPusCfsucaAfaGf CAACAUCCGUUAUUAC 3449 801658.2 1535093.1 AfUfuacuuugagaL96 1535094.1 UfaauaAfcGfgaug UUUGAGA ususg AD- A- 2017 asgsaca(Uhd)UfuGf A- 2018 VPusGfsuagAfuUf UGAGACAUUUGUCCUA 3450 801676.2 1535129.1 UfCfcuaaucuacaL96 1535130.1 AfggacAfaAfuguc AUCUACG uscsa AD- A- 2019 usgscca(Chd)UfgAf A- 2020 VPusCfsaguAfcUf GUUGCCACUGAAGAAA 3451 799683.1 1531160.1 AfGfaaaguacugaL96 1531161.1 UfucuuCfaGfuggc GUACUGA asasc AD- A- 2021 uscsauc(Uhd)UfuUf A- 2022 VPusAfscaaUfcCf UGUCAUCUUUUCACAG 3452 800486.1 1532749.1 CfAfcaggauuguaL96 1532750.1 UfgugaAfaAfgaug GAUUGUA ascsa AD- A- 2023 csgsgac(Uhd)UfgGf A- 2024 VPusGfsagaUfaGf GUCGGACUUGGUUACC 3453 798672.1 1529207.1 UfUfaccuaucucaL96 1529208.1 GfuaacCfaAfgucc UAUCUCU gsasc AD- A- 2025 csuscuu(Uhd)CfcAf A- 2026 VPusAfsguaAfuCf CUCUCUUUCCAUGUAG 3454 802145.2 1536067.1 UfGfuagauuacuaL9 1536068.1 UfacauGfgAfaaga AUUACUG 6 gsasg AD- A- 2027 ascsaac(Uhd)UfuCf A- 2028 VPusAfsgcaAfaUf AAACAACUUUCACUAA 3455 801540.2 1534857.1 AfCfuaauuugcuaL96 1534858.1 UfagugAfaAfguug UUUGCUU USUSU AD- A- 2029 usascaa(Chd)AfuCf A- 2030 VPusAfsaguAfaUf UUUACAACAUCCGUUA 3456 801654.2 1535085.1 CfGfuuauuacuuaL96 1535086.1 AfacggAfuGfuugu UUACUUU asasa AD- A- 2031 asasugu(Chd)GfgAf A- 2032 VPusAfsgguAfaCf AUAAUGUCGGACUUG 3457 798667.1 1529197.1 CfUfugguuaccuaL96 1529198.1 CfaaguCfcGfacau GUUACCUA usasu AD- A- 2033 ascsaac(Ahd)UfcCf A- 2034 VPusAfsaagUfaAf UUACAACAUCCGUUAU 3458 801655.2 1535087.1 GfUfuauuacuuuaL9 1535088.1 UfaacgGfaUfguug UACUUUG 6 usasa AD- A- 2035 csusucu(Uhd)AfgCf A- 2036 VPusGfsccuAfaAf GCCUUCUUAGCCUUGU 3459 795826.1 1523683.1 CfUfuguuuaggcaL96 1523684.1 CfaaggCfuAfagaa UUAGGCU gsgsc AD- A- 2037 ascsaca(Ghd)GfuAf A- 2038 VPusAfsaacUfaCf CUACACAGGUAGAAUG 3460 801490.2 1534757.1 GfAfauguaguuuaL9 1534758.1 AfuucuAfcCfugug UAGUUUU 6 usasg AD- A- 2039 csusgaa(Chd)CfuAf A- 2040 VPusAfsucgGfaAf GGCUGAACCUAUGAAU 3461 797964.1 1527831.1 UfGfaauuccgauaL96 1527832.1 UfucauAfgGfuuca UCCGAUG gscsc AD- A- 2041 asusucu(Uhd)UfaAf A- 2042 VPusUfscuuAfcGf UGAUUCUUUAAGAAU 3462 800389.2 1532555.1 GfAfaucguaagaaL96 1532556.1 AfuucuUfaAfagaa CGUAAGAG uscsa AD- A- 2043 gsasuuc(Uhd)UfuAf A- 2044 VPusCfsuuaCfgAf AUGAUUCUUUAAGAA 3463 800388.2 1532553.1 AfGfaaucguaagaL96 1532554.1 UfucuuAfaAfgaau UCGUAAGA csasu AD- A- 2045 gsusuuc(Ahd)GfgAf A- 2046 VPusCfsaagUfaGf UUGUUUCAGGAAUGU 3464 802070.2 1535917.1 AfUfgucuacuugaL96 1535918.1 AfcauuCfcUfgaaa CUACUUGU csasa AD- A- 2047 usasuag(Ahd)AfaCf A- 2048 VPusCfsauaAfaUf CCUAUAGAAACAAAGA 3465 801601.2 1534979.1 AfAfagauuuaugaL96 1534980.1 CfuuugUfuUfcua UUUAUGG uasgsg AD- A- 2049 ususaca(Ahd)CfaUf A- 2050 VPusAfsguaAfuAf UUUUACAACAUCCGUU 3466 801653.1 1535083.1 CfCfguuauuacuaL96 1535084.1 AfcggaUfgUfugua AUUACUU asasa AD- A- 2051 ususuca(Ghd)GfaAf A- 2052 VPusAfscaaGfuAf UGUUUCAGGAAUGUC 3467 802071.2 1535919.1 UfGfucuacuuguaL96 1535920.1 GfacauUfcCfugaa UACUUGUG ascsa AD- A- 2053 gsasuaa(Uhd)AfgUf A- 2054 VPusGfsaguUfuAf CUGAUAAUAGUCUCU 3468 800968.2 1533713.1 CfUfcuuaaacucaL96 1533714.1 AfgagaCfuAfuuau UAAACUCU csasg AD- A- 2055 asgsagg(Uhd)UfaUf A- 2056 VPusCfsaaaAfuAf ACAGAGGUUAUUCUA 3469 800667.2 1533111.1 UfCfuauauuuugaL96 1533112.1 UfagaaUfaAfccuc UAUUUUGA usgsu AD- A- 2057 uscsaca(Ahd)CfcAfC A- 2058 VPusCfscguUfuUf CAUCACAACCACACUAA 3470 800008.2 1531795.1 fAfcuaaaacggaL96 1531796.1 AfguguGfgUfugu AACGGA gasusg AD- A- 2059 ascsaca(Ahd)UfuUf A- 2060 VPusAfsugcUfaAf CAACACAAUUUCUUCU 3471 802016.2 1535809.1 CfUfucuuagcauaL96 1535810.1 GfaagaAfaUfugug UAGCAUU ususg AD- A- 2061 uscsauc(Chd)UfgGf A- 2062 VPusCfsaacUfgAf GUUCAUCCUGGAAGU 3472 799549.1 1530912.1 AfAfguucaguugaL96 1530913.1 AfcuucCfaGfgaug UCAGUUGA asasc AD- A- 2063 ususgca(Uhd)CfaGf A- 2064 VPusAfsuaaAfuUf CAUUGCAUCAGAACCA 3473 800706.2 1533189.1 AfAfccaauuuauaL96 1533190.1 GfguucUfgAfugca AUUUAUA asusg AD- A- 2065 ususcau(Chd)UfuAf A- 2066 VPusUfsucaAfaUf CAUUCAUCUUAGGCUA 3474 801746.2 1535269.1 GfGfcuauuugaaaL96 1535270.1 AfgccuAfaGfauga UUUGAAC asusg AD- A- 2067 gsasuuc(Uhd)UfuAf A- 2068 VPusCfsuaaGfaUf AAGAUUCUUUAUACCA 3475 801721.2 1535219.1 UfAfccaucuuagaL96 1535220.1 GfguauAfaAfgaau UCUUAGG csusu AD- A- 2069 asusaau(Chd)GfcUf A- 2070 VPusGfsuaaUfaAf UUAUAAUCGCUGAACU 3476 802205.2 1536187.1 GfAfacuuauuacaL96 1536188.1 GfuucaGfcGfauua UAUUACA usasa AD- A- 2071 asusuug(Uhd)CfcUf A- 2072 VPusAfsuacGfuAf ACAUUUGUCCUAAUCU 3477 801680.2 1535137.1 AfAfucuacguauaL96 1535138.1 GfauuaGfgAfcaaa ACGUAUA usgsu AD- A- 2073 ususuua(Chd)AfuCf A- 2074 VPusAfsugaCfaAf ACUUUUACAUCUGCCU 3478 800470.1 1532717.1 UfGfccuugucauaL96 1532718.1 GfgcagAfuGfuaaa UGUCAUC asgsu AD- A- 2075 ascsauu(Uhd)GfuCf A- 2076 VPusAfscguAfgAf AGACAUUUGUCCUAAU 3479 801678.2 1535133.1 CfUfaaucuacguaL96 1535134.1 UfuaggAfcAfaaug CUACGUA uscsu AD- A- 2077 usgsuuu(Ahd)GfuCf A- 2078 VPusAfsgcgAfaAf AUUGUUUAGUCAUCC 3480 801022.2 1533821.1 AfUfccuuucgcuaL96 1533822.1 GfgaugAfcUfaaac UUUCGCUG asasu AD- A- 2079 uscsucc(Uhd)UfaAf A- 2080 VPusAfsucaUfaGf CUUCUCCUUAAAAUUC 3481 801309.2 1534395.1 AfAfuucuaugauaL96 1534396.1 AfauuuUfaAfggag UAUGAUG asasg AD- A- 2081 ascsagg(Ahd)UfuGf A- 2082 VPusAfsagaCfuAf UCACAGGAUUGUAAU 3482 800496.2 1532769.1 UfAfauuagucuuaL96 1532770.1 AfuuacAfaUfccug UAGUCUUG usgsa AD- A- 2083 usasggu(Uhd)CfaUf A- 2084 VPusGfsccuAfaGf CUUAGGUUCAUUCAUC 3483 801738.2 1535253.1 UfCfaucuuaggcaL96 1535254.1 AfugaaUfgAfaccu UUAGGCU asasg AD- A- 2085 asascaa(Chd)UfuUf A- 2086 VPusGfscaaAfuUf AAAACAACUUUCACUA 3484 801539.2 1534855.1 CfAfcuaauuugcaL96 1534856.1 AfgugaAfaGfuugu AUUUGCU ususu AD- A- 2087 asasgcc(Uhd)UfuGf A- 2088 VPusGfsauaCfuAf UCAAGCCUUUGAUAU 3485 799010.2 1529846.1 AfUfauuaguaucaL96 1529847.1 AfuaucAfaAfggcu UAGUAUCA usgsa AD- A- 2089 csusuuc(Uhd)UfcUf A- 2090 VPusAfsgggAfuAf GCCUUUCUUCUUUCAU 3486 800850.2 1533477.1 UfUfcauaucccuaL96 1533478.1 UfgaaaGfaAfgaaa AUCCCUU gsgsc AD- A- 2091 uscsaca(Ghd)GfaUf A- 2092 VPusGfsacuAfaUf UUUCACAGGAUUGUA 3487 800494.2 1532765.1 UfGfuaauuagucaL96 1532766.1 UfacaaUfcCfugug AUUAGUCU asasa AD- A- 2093 ususgcc(Chd)UfuAf A- 2094 VPusAfscuaAfcAf UUUUGCCCUUAUGAA 3488 798614.1 1529091.1 UfGfaauguuaguaL96 1529092.1 UfucauAfaGfggca UGUUAGUC asasa AD- A- 2095 csasuca(Ghd)AfaCfC A- 2096 VPusCfsauaUfaAf UGCAUCAGAACCAAUU 3489 800709.2 1533195.1 fAfauuuauaugaL96 1533196.1 AfuuggUfuCfugau UAUAUGU gscsa AD- A- 2097 asusuca(Ahd)UfcUf A- 2098 VPusGfsaaaUfaAf GAAUUCAAUCUACCGU 3490 801888.2 1535553.1 AfCfcguuauuucaL96 1535554.1 CfgguaGfaUfugaa UAUUUCA USUSC AD- A- 2099 ususucg(Chd)UfgUf A- 2100 VPusCfsaacUfuUf CCUUUCGCUGUAAGCA 3491 801035.2 1533847.1 AfAfgcaaaguugaL96 1533848.1 GfcuuaCfaGfcgaa AAGUUGA asgsg AD- A- 2101 asusugu(Uhd)UfaGf A- 2102 VPusCfsgaaAfgGf GUAUUGUUUAGUCAU 3492 801020.2 1533817.1 UfCfauccuuucgaL96 1533818.1 AfugacUfaAfacaa CCUUUCGC usasc AD- A- 2103 gsasgac(Ahd)UfuUf A- 2104 VPusUfsagaUfuAf UUGAGACAUUUGUCC 3493 801675.2 1535127.1 GfUfccuaaucuaaL96 1535128.1 GfgacaAfaUfgucu UAAUCUAC csasa AD- A- 2105 ususgcc(Ahd)AfcUf A- 2106 VPusGfscaaGfaGf UCUUGCCAACUUGCUC 3494 801228.2 1534233.1 UfGfcucucuugcaL96 1534234.1 AfgcaaGfuUfggca UCUUGCC asgsa AD- A- 2107 asusgua(Uhd)AfuUf A- 2108 VPusUfscacUfaGf GGAUGUAUAUUUGAC 3495 798984.1 1529794.1 UfGfaccuagugaaL96 1529795.1 GfucaaAfuAfuaca CUAGUGAC uscsc AD- A- 2109 csascag(Ghd)AfuUf A- 2110 VPusAfsgacUfaAf UUCACAGGAUUGUAA 3496 800495.2 1532767.1 GfUfaauuagucuaL96 1532768.1 UfuacaAfuCfcugu UUAGUCUU gsasa AD- A- 2111 gsasugu(Uhd)UfgAf A- 2112 VPusAfscacGfaAf AAGAUGUUUGACAGG 3497 801957.2 1535691.1 CfAfgguucguguaL96 1535692.1 CfcuguCfaAfacau UUCGUGUG csusu AD- A- 2113 usasgcu(Ghd)UfaGf A- 2114 VPusAfsaacUfaGf AUUAGCUGUAGACAUC 3498 801399.2 1534575.1 AfCfaucuaguuuaL96 1534576.1 AfugucUfaCfagcu UAGUUUU asasu AD- A- 2115 usascac(Ahd)GfgUf A- 2116 VPusAfsacuAfcAf GCUACACAGGUAGAAU 3499 801489.2 1534755.1 AfGfaauguaguuaL96 1534756.1 UfucuaCfcUfgugu GUAGUUU asgsc AD- A- 2117 asgsucu(Chd)UfuAf A- 2118 VPusAfscaaAfaGf AUAGUCUCUUAAACUC 3500 800974.2 1533725.1 AfAfcucuuuuguaL96 1533726.1 AfguuuAfaGfagac UUUUGUC usasu AD- A- 2119 asuscac(Ahd)AfcCfA A- 2120 VPusCfsguuUfuAf CCAUCACAACCACACUA 3501 800007.2 1531793.1 fCfacuaaaacgaL96 1531794.1 GfugugGfuUfgug AAACGG ausgsg AD- A- 2121 csasuuu(Ghd)UfcCf A- 2122 VPusUfsacgUfaGf GACAUUUGUCCUAAUC 3502 801679.2 1535135.1 UfAfaucuacguaaL96 1535136.1 AfuuagGfaCfaaau UACGUAU gsusc AD- A- 2123 csusgcc(Ahd)AfgUf A- 2124 VPusAfscucUfaUf UGCUGCCAAGUUAACA 3503 798031.1 1527964.1 UfAfacauagaguaL96 1527965.1 GfuuaaCfuUfggca UAGAGUC gscsa AD- A- 2125 asusuag(Chd)UfgUf A- 2126 VPusAfscuaGfaUf GCAUUAGCUGUAGACA 3504 801397.2 1534571.1 AfGfacaucuaguaL96 1534572.1 GfucuaCfaGfcuaa UCUAGUU usgsc AD- A- 2127 gsuscuc(Uhd)UfaAf A- 2128 VPusGfsacaAfaAf UAGUCUCUUAAACUCU 3505 800975.2 1533727.1 AfCfucuuuugucaL96 1533728.1 GfaguuUfaAfgaga UUUGUCA csusa AD- A- 2129 gsascau(Uhd)UfgUf A- 2130 VPusCfsguaGfaUf GAGACAUUUGUCCUAA 3506 801677.2 1535131.1 CfCfuaaucuacgaL96 1535132.1 UfaggaCfaAfaugu UCUACGU csusc AD- A- 2131 ususcuu(Uhd)AfuAf A- 2132 VPusAfsccuAfaGf GAUUCUUUAUACCAUC 3507 801723.2 1535223.1 CfCfaucuuagguaL96 1535224.1 AfugguAfuAfaaga UUAGGUU asusc AD- A- 2133 csascag(Ghd)UfaGf A- 2134 VPusAfsaaaCfuAf UACACAGGUAGAAUGU 3508 801491.2 1534759.1 AfAfuguaguuuuaL96 1534760.1 CfauucUfaCfcugu AGUUUUA gsusa AD- A- 2135 asusgua(Ghd)AfuUf A- 2136 VPusGfsuacAfaAf CCAUGUAGAUUACUGU 3509 802153.2 1536083.1 AfCfuguuuguacaL96 1536084.1 CfaguaAfuCfuaca UUGUACU usgsg AD- A- 2137 uscsacu(Uhd)GfuAf A- 2138 VPusAfscggGfaUf CAUCACUUGUAUACAA 3510 801140.2 1534057.1 UfAfcaaucccguaL96 1534058.1 UfguauAfcAfagug UCCCGUG asusg AD- A- 2139 asusuca(Uhd)CfuUf A- 2140 VPusUfscaaAfuAf UCAUUCAUCUUAGGCU 3511 801745.2 1535267.1 AfGfgcuauuugaaL96 1535268.1 GfccuaAfgAfugaa AUUUGAA usgsa AD- A- 2141 csasuuc(Ahd)UfcUf A- 2142 VPusCfsaaaUfaGf UUCAUUCAUCUUAGGC 3512 801744.2 1535265.1 UfAfggcuauuugaL96 1535266.1 CfcuaaGfaUfgaau UAUUUGA gsasa AD- A- 2143 asgsagc(Uhd)UfaUf A- 2144 VPusGfscuuAfuAf AAAGAGCUUAUUAAG 3513 802106.2 1535989.1 UfAfaguauaagcaL96 1535990.1 CfuuaaUfaAfgcuc UAUAAGCU ususu AD- A- 2145 usgsaug(Ahd)UfuCf A- 2146 VPusCfsgauUfcUf ACUGAUGAUUCUUUA 3514 800384.2 1532545.1 UfUfuaagaaucgaL96 1532546.1 UfaaagAfaUfcauc AGAAUCGU asgsu AD- A- 2147 csasaca(Ghd)AfuGf A- 2148 VPusAfsgacGfgUf UUCAACAGAUGUUAGA 3515 796041.1 1524103.1 UfUfagaccgucuaL96 1524104.1 CfuaacAfuCfuguu CCGUCUU gsasa

TABLE 4B Exemplary Human SCN9A Unmodified Single Strands and Duplex Sequences. Column 1 indicates duplex name and the number following the decimal point in a duplex name merely refers to a batch production number. Column 2 indicates the sense sequence name. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the unmodified sequence of a sense strand suitable for use in a duplex described herein. Column 5 provides the position in the target mRNA (NM_001365536.1) of the sense strand of Column 4. Column 6 indicates the antisense sequence name. Column 7 indicates the sequence ID for the sequence of column 8. Column 8 provides the sequence of an antisense strand suitable for use in a duplex described herein, without specifying chemical modifications. Column 9 indicates the position in the target mRNA (NM_001365536.1) that is complementary to the antisense strand of Column 8. mRNA target Anti Seq ID mRNA target Sense Seq ID range in sense NO: antisense range in Duplex sequence NO: Sense sequence NM_00136 sequence (anti sequence NM_0013 Name name (sense) (5′-3′) 5536.1 name sense) (5′-3′) 65536.1 AD- A- 2149 UUUGUAGAUCUUGCAAUU 2531-2551 A- 2150 UGUAAUUGCAAGAUC 2529-2551 796825.1 1525636.1 ACA 1257916.1 UACAAAAG AD- A- 2151 UUCUGUGUAGGAGAAUUC 824-844 A- 2152 UGUGAAUUCUCCUACA 822-844 795366.1 1522818.1 ACA 1522819.1 CAGAAGC AD- A- 2153 AUGUGAAACAAACCUUAC 3300-3320 A- 2154 UACGUAAGGUUUGUU 3298-3320 797565.2 1527044.1 GUA 1527045.1 UCACAUAA AD- A- 2155 UGUAGGAGAAUUCACUUU 829-849 A- 2156 UGAAAAGUGAAUUCUC 827-849 795371.1 1522828.1 UCA 1522829.1 CUACACA AD- A- 2157 UAUGUGAAACAAACCUUA 3299-3319 A- 2158 UCGUAAGGUUUGUUU 3297-3319 797564.2 1527042.1 CGA 1527043.1 CACAUAAU AD- A- 2159 AGCAUAAAUGUUUUCGAA 1113-1133 A- 2160 UAUUUCGAAAACAUU 1111-1133 795634.2 1523299.1 AUA 1523300.1 UAUGCUUC AD- A- 2161 GAUCUUCUUUGUCGUAGU 1435-1455 A- 2162 UUCACUACGACAAAGA 1433-1455 795913.1 1523849.1 GAA 1523850.1 AGAUCAU AD- A- 2163 GGCGUUGUAGUUCCUAUC 2301-2321 A- 2164 UGAGAUAGGAACUACA 2299-2321 796618.1 1525247.1 UCA 1525248.1 ACGCCUU AD- A- 2165 AUCUUCUUUGUCGUAGUG 1436-1456 A- 2166 UAUCACUACGACAAAG 1434-1456 795914.1 1523851.1 AUA 1523852.1 AAGAUCA AD- A- 2167 UGGUUUCAGCACAGAUUC 1243-1263 A- 2168 UCUGAAUCUGUGCUG 1241-1263 795739.1 1523509.1 AGA 1523510.1 AAACCACA AD- A- 2169 UGUCGAGUACACUUUUAC 760-780 A- 2170 UCAGUAAAAGUGUACU 758-780 795305.1 1522697.1 UGA 1522698.1 CGACAUU AD- A- 2171 AAGCAGAAGAUCUGAAUA 3375-3395 A- 2172 UAGUAUUCAGAUCUU 3373-3395 797636.2 1527186.1 CUA 1527187.1 CUGCUUGU AD- A- 2173 CAAGUGUUCCUACUGUCA 9104-9124 A- 2174 UCAUGACAGUAGGAAC 9102-9124 802471.2 1536717.1 UGA 1536718.1 ACUUGAA AD- A- 2175 AUGCUGAGAAAUUGUCGA 1785-1805 A- 2176 UUUUCGACAAUUUCUC 1783-1805 796209.1 1524439.1 AAA 1524440.1 AGCAUCU AD- A- 2177 AUGUUUCUAGCUGAUUU 5075-5095 A- 2178 UAUCAAAUCAGCUAGA 5073-5095 799223.1 1530270.1 GAUA 1530271.1 AACAUAC AD- A- 2179 GAGAUGGAUUCUCUUCGU 5861-5881 A- 2180 UGAACGAAGAGAAUCC 5859-5881 799938.1 1531655.1 UCA 1531656.1 AUCUCCC AD- A- 2181 UUGUGACUUUAAGUUUA 2742-2762 A- 2182 UCACUAAACUUAAAGU 2740-2762 797036.1 1526036.1 GUGA 1526037.1 CACAAUA AD- A- 2183 AUGAUCUUCUUUGUCGUA 1433-1453 A- 2184 UACUACGACAAAGAAG 1431-1453 795911.1 1523845.1 GUA 1523846.1 AUCAUGU AD- A- 2185 AAGGGAAAACAAUCUUCC 576-596 A- 2186 UACGGAAGAUUGUUU 574-596 795132.1 1522351.1 GUA 1522352.1 UCCCUUUG AD- A- 2187 CUUCUGAAACAUCCAAACU 1683-1703 A- 2188 UCAGUUUGGAUGUUU 1681-1703 796138.1 1524297.1 GA 1524298.1 CAGAAGAA AD- A- 2189 UUGCUAUAGGAAAUUUGG 2625-2645 A- 2190 UGACCAAAUUUCCUAU 2623-2645 796919.1 1525802.1 UCA 1525803.1 AGCAAGU AD- A- 2191 UAUUGUGACUUUAAGUU 2740-2760 A- 2192 UCUAAACUUAAAGUCA 2738-2760 797034.1 1526032.1 UAGA 1526033.1 CAAUAAG AD- A- 2193 UUGGCAGAAACCCUGAUU 1296-1316 A- 2194 UAUAAUCAGGGUUUC 1294-1316 795774.1 1523579.1 AUA 1523580.1 UGCCAAUU AD- A- 2195 ACAUGAUCUUCUUUGUCG 1431-1451 A- 2196 UUACGACAAAGAAGAU 1429-1451 795909.1 1523841.1 UAA 1523842.1 CAUGUAG AD- A- 2197 AGCUUGAAGUAAAAUUAG 8687-8707 A- 2198 UGUCUAAUUUUACUU 8685-8707 802123.1 1536023.1 ACA 1536024.1 CAAGCUUA AD- A- 2199 UCCAAAUCGUUCCGAAUG 4390-4410 A- 2200 UAACAUUCGGAACGAU 4388-4410 798588.2 1529045.1 UUA 1529046.1 UUGGAAC AD- A- 2201 AUCUGAGACUGAAUUUGC 1993-2013 A- 2202 UCGGCAAAUUCAGUCU 1991-2013 796396.1 1524811.1 CGA 1524812.1 CAGAUCC AD- A- 2203 GCGUUGUAGUUCCUAUCU 2302-2322 A- 2204 UGGAGAUAGGAACUAC 2300-2322 796619.1 1525249.1 CCA 1525250.1 AACGCCU AD- A- 2205 UAUAUUUUACAACAUCCG 8022-8042 A- 2206 UAACGGAUGUUGUAA 8020-8042 801647.1 1535071.1 UUA 1535072.1 AAUAUAUC AD- A- 2207 AUGUCGAGUACACUUUUA 759-779 A- 2208 UAGUAAAAGUGUACUC 757-779 795304.1 1522695.1 CUA 1522696.1 GACAUUU AD- A- 2209 UGAUAGUUACCUAGUUUG 9226-9246 A- 2210 UUGCAAACUAGGUAAC 9224-9246 802553.1 1536879.1 CAA 1536880.1 UAUCAAA AD- A- 2211 GACUUACCUUUAGAGUAU 6944-6964 A- 2212 UCAAUACUCUAAAGGU 6942-6964 800819.1 1533415.1 UGA 1533416.1 AAGUCUU AD- A- 2213 CUAAAUUAUGGAAGUAAU 7468-7488 A- 2214 UAGAUUACUUCCAUAA 7466-7488 801263.1 1534303.1 CUA 1534304.1 UUUAGGA AD- A- 2215 AGUCAAGUUCCAAAUCGU 4382-4402 A- 2216 UGAACGAUUUGGAAC 4380-4402 798580.1 1529029.1 UCA 1529030.1 UUGACUUG AD- A- 2217 UGAUCUUCUUUGUCGUAG 1434-1454 A- 2218 UCACUACGACAAAGAA 1432-1454 795912.1 1523847.1 UGA 1523848.1 GAUCAUG AD- A- 2219 GUUUGAACACAAAUCUUU 9174-9194 A- 2220 UCGAAAGAUUUGUGU 9172-9194 802503.1 1536779.1 CGA 1536780.1 UCAAACCU AD- A- 2221 AAGUUCCAAAUCGUUCCG 4386-4406 A- 2222 UUUCGGAACGAUUUG 4384-4406 798584.2 1529037.1 AAA 1529038.1 GAACUUGA AD- A- 2223 UGUAGAUCUUGCAAUUAC 2533-2553 A- 2224 UUGGUAAUUGCAAGA 2531-2553 796827.1 1525638.1 CAA 1257918.1 UCUACAAA AD- A- 2225 CAUGAUCUUCUUUGUCGU 1432-1452 A- 2226 UCUACGACAAAGAAGA 1430-1452 795910.1 1523843.1 AGA 1523844.1 UCAUGUA AD- A- 2227 UUGAUAGUUACCUAGUUU 9225-9245 A- 2228 UGCAAACUAGGUAACU 9223-9245 802552.1 1536877.1 GCA 1536878.1 AUCAAAA AD- A- 2229 CACCUUCUCCUUAAAAUU 7527-7547 A- 2230 UAGAAUUUUAAGGAG 7525-7547 801304.1 1534385.1 CUA 1534386.1 AAGGUGAC AD- A- 2231 CUGAUUUCCUAAGAAAGG 6396-6416 A- 2232 UCACCUUUCUUAGGAA 6394-6416 800334.1 1532445.1 UGA 1532446.1 AUCAGAG AD- A- 2233 UGAGACUGACACAUUGUA 9700-9720 A- 2234 UAUUACAAUGUGUCA 9698-9720 802946.1 1537662.1 AUA 1537663.1 GUCUCAAG AD- A- 2235 CUGAAUAUACAAGUAUUA 1632-1652 A- 2236 UCCUAAUACUUGUAUA 1630-1652 796087.1 1524195.1 GGA 1524196.1 UUCAGCC AD- A- 2237 CAACCCAAAAUACUUAGCA 9298-9318 A- 2238 UAUGCUAAGUAUUUU 9296-9318 802625.2 1537023.1 UA 1537024.1 GGGUUGUG AD- A- 2239 CUGAUAAUAGUCUCUUAA 7151-7171 A- 2240 UGUUUAAGAGACUAU 7149-7171 800966.1 1533709.1 ACA 1533710.1 UAUCAGUA AD- A- 2241 UUUGUCGUAGUGAUUUU 1442-1462 A- 2242 UAGGAAAAUCACUACG 1440-1462 795920.1 1523863.1 CCUA 1523864.1 ACAAAGA AD- A- 2243 UGAAUAUACAAGUAUUAG 1633-1653 A- 2244 UUCCUAAUACUUGUA 1631-1653 796088.1 1524197.1 GAA 1524198.1 UAUUCAGC AD- A- 2245 AGAUGGAUUCUCUUCGUU 5862-5882 A- 2246 UUGAACGAAGAGAAUC 5860-5882 799939.1 1531657.1 CAA 1531658.1 CAUCUCC AD- A- 2247 AAUAUCAUAAAGCUGUUU 9589-9609 A- 2248 UGUAAACAGCUUUAU 9587-9609 802853.2 1537477.1 ACA 1537478.1 GAUAUUCA AD- A- 2249 UCUUUAUACCAUCUUAGG 8099-8119 A- 2250 UAACCUAAGAUGGUAU 8097-8119 801724.1 1535225.1 UUA 1535226.1 AAAGAAU AD- A- 2251 GCAAAGGUCACAAUUUCC 3438-3458 A- 2252 UGAGGAAAUUGUGAC 3436-3458 797699.1 1527312.1 UCA 1527313.1 CUUUGCUC AD- A- 2253 AGUCACCACUCAGCAUUCG 1899-1919 A- 2254 UACGAAUGCUGAGUG 1897-1919 796304.1 1524627.1 UA 1524628.1 GUGACUGA AD- A- 2255 UGCUAUAGGAAAUUUGGU 2626-2646 A- 2256 UAGACCAAAUUUCCUA 2624-2646 796920.1 1525804.1 CUA 1525805.1 UAGCAAG AD- A- 2257 GACAGAGAUGAUGAUUUA 6059-6079 A- 2258 UAGUAAAUCAUCAUCU 6057-6079 800110.1 1531997.1 CUA 1531998.1 CUGUCUC AD- A- 2259 AAGUCAAGUUCCAAAUCG 4381-4401 A- 2260 UAACGAUUUGGAACU 4379-4401 798579.1 1529027.1 UUA 1529028.1 UGACUUGC AD- A- 2261 UAGGCUAAUGACCCAAGA 1363-1383 A- 2262 UAAUCUUGGGUCAUU 1361-1383 795841.1 1523713.1 UUA 1523714.1 AGCCUAAA AD- A- 2263 AAGAGCUUAUUAAGUAUA 8669-8689 A- 2264 UCUUAUACUUAAUAA 8667-8689 802105.2 1535987.1 AGA 1535988.1 GCUCUUUC AD- A- 2265 UGGAAUAUUCUACUUUGU 5503-5523 A- 2266 UUAACAAAGUAGAAUA 5501-5523 799594.1 1531002.1 UAA 1531003.1 UUCCAAC AD- A- 2267 AUGUACAGAGGUUAUUCU 6778-6798 A- 2268 UAUAGAAUAACCUCUG 6776-6798 800661.1 1533099.1 AUA 1533100.1 UACAUUG AD- A- 2269 AUCGUAAGAGAACUCUGU 6462-6482 A- 2270 UCUACAGAGUUCUCUU 6460-6482 800400.1 1532577.1 AGA 1532578.1 ACGAUUC AD- A- 2271 CAUCUGUUGGAAUAUUCU 5496-5516 A- 2272 UGUAGAAUAUUCCAAC 5494-5516 799587.1 1530988.1 ACA 1530989.1 AGAUGGG AD- A- 2273 GUCUUUACUGGAAUCUUU 2642-2662 A- 2274 UGCAAAGAUUCCAGUA 2640-2662 796936.1 1525836.1 GCA 1525837.1 AAGACCA AD- A- 2275 CAACACAAUUUCUUCUUA 8498-8518 A- 2276 UGCUAAGAAGAAAUU 8496-8518 802014.1 1535805.1 GCA 1535806.1 GUGUUGUU AD- A- 2277 UGGAUUCUCUUCGUUCAC 5865-5885 A- 2278 UCUGUGAACGAAGAGA 5863-5885 799942.1 1531663.1 AGA 1531664.1 AUCCAUC AD- A- 2279 GUAUGUUUCUAGCUGAU 5073-5093 A- 2280 UCAAAUCAGCUAGAAA 5071-5093 799221.1 1530266.1 UUGA 1530267.1 CAUACCU AD- A- 2281 CCUUCCUGAUAUGCAGUU 7247-7267 A- 2282 UCUAACUGCAUAUCAG 7245-7267 801062.1 1533901.1 AGA 1533902.1 GAAGGAU AD- A- 2283 GGAGAUGGAUUCUCUUCG 5860-5880 A- 2284 UAACGAAGAGAAUCCA 5858-5880 799937.1 1531653.1 UUA 1531654.1 UCUCCCC AD- A- 2285 GUAGAAAACUUUUACAUC 6547-6567 A- 2286 UCAGAUGUAAAAGUU 6545-6567 800461.1 1532699.1 UGA 1532700.1 UUCUACAU AD- A- 2287 AGCGUGCUUAUAGACGUU 5988-6008 A- 2288 UGUAACGUCUAUAAGC 5986-6008 800058.1 1531895.1 ACA 1531896.1 ACGCUGA AD- A- 2289 GUUUCUAGCUGAUUUGA 5077-5097 A- 2290 UCAAUCAAAUCAGCUA 5075-5097 799225.1 1530274.1 UUGA 1530275.1 GAAACAU AD- A- 2291 GCCCAAAAUACUGAUAAU 7141-7161 A- 2292 UCUAUUAUCAGUAUU 7139-7161 800956.1 1533689.1 AGA 1533690.1 UUGGGCAG AD- A- 2293 UUUGUCCUAAUCUACGUA 8056-8076 A- 2294 UUAUACGUAGAUUAG 8054-8076 801681.2 1535139.1 UAA 1535140.1 GACAAAUG AD- A- 2295 UAAUCGCUGAACUUAUUA 8787-8807 A- 2296 UUGUAAUAAGUUCAG 8785-8807 802206.2 1536189.1 CAA 1536190.1 CGAUUAUA AD- A- 2297 UUUGAAUUCAAUCUACCG 8327-8347 A- 2298 UAACGGUAGAUUGAA 8325-8347 801883.2 1535543.1 UUA 1535544.1 UUCAAAUU AD- A- 2299 CUCUUUUGAGGAAGUCUA 6326-6346 A- 2300 UCAUAGACUUCCUCAA 6324-6346 800273.2 1532323.1 UGA 1532324.1 AAGAGUU AD- A- 2301 AGCUGAUUUGAUUGAAAC 5083-5103 A- 2302 UACGUUUCAAUCAAAU 5081-5103 799231.2 1530286.1 GUA 1530287.1 CAGCUAG AD- A- 2303 CUUUAUACCAUCUUAGGU 8100-8120 A- 2304 UGAACCUAAGAUGGUA 8098-8120 801725.1 1535227.1 UCA 1535228.1 UAAAGAA AD- A- 2305 UUGCAAGCCUCUUAUGUG 243-263 A- 2306 UCUCACAUAAGAGGCU 241-263 794914.1 1521918.1 AGA 1521919.1 UGCAACC AD- A- 2307 UUAUUGCAUCACUUGUAU 7317-7337 A- 2308 UGUAUACAAGUGAUG 7315-7337 801132.1 1534041.1 ACA 1534042.1 CAAUAAAU AD- A- 2309 UUUCACAGGAUUGUAAUU 6578-6598 A- 2310 UCUAAUUACAAUCCUG 6576-6598 800492.2 1532761.1 AGA 1532762.1 UGAAAAG AD- A- 2311 CUUUUCACAGGAUUGUAA 6576-6596 A- 2312 UAAUUACAAUCCUGUG 6574-6596 800490.1 1532757.1 UUA 1532758.1 AAAAGAU AD- A- 2313 CUGUAGGAAUUAUUGAUU 6476-6496 A- 2314 UAUAAUCAAUAAUUCC 6474-6496 800414.2 1532605.1 AUA 1532606.1 UACAGAG AD- A- 2315 UUCCUGAUAUGCAGUUAG 7249-7269 A- 2316 UAACUAACUGCAUAUC 7247-7269 801064.1 1533905.1 UUA 1533906.1 AGGAAGG AD- A- 2317 GCAAGUCAAGUUCCAAAU 4379-4399 A- 2318 UCGAUUUGGAACUUG 4377-4399 798577.1 1529023.1 CGA 1529024.1 ACUUGCAG AD- A- 2319 GGAAGAAAGGUUCAUGUC 5887-5907 A- 2320 UCAGACAUGAACCUUU 5885-5907 799959.1 1531697.1 UGA 1531698.1 CUUCCAU AD- A- 2321 AUCUAGGGCUAAAGAUUC 8083-8103 A- 2322 UAAGAAUCUUUAGCCC 8081-8103 801708.2 1535193.1 UUA 1535194.1 UAGAUUG AD- A- 2323 UAGCUGAUUUGAUUGAAA 5082-5102 A- 2324 UCGUUUCAAUCAAAUC 5080-5102 799230.2 1530284.1 CGA 1530285.1 AGCUAGA AD- A- 2325 CUUCCUGAUAUGCAGUUA 7248-7268 A- 2326 UACUAACUGCAUAUCA 7246-7268 801063.1 1533903.1 GUA 1533904.1 GGAAGGA AD- A- 2327 ACUGAUGAUUCUUUAAGA 6444-6464 A- 2328 UAUUCUUAAAGAAUCA 6442-6464 800382.2 1532541.1 AUA 1532542.1 UCAGUGC AD- A- 2329 AGACGUUACCGCUUAAGG 5999-6019 A- 2330 UUGCCUUAAGCGGUAA 5997-6019 800069.1 1531917.1 CAA 1531918.1 CGUCUAU AD- A- 2331 UCGUGGCUCCUUGUUUUC 1915-1935 A- 2332 UCAGAAAACAAGGAGC 1913-1935 796318.1 1524655.1 UGA 1524656.1 CACGAAU AD- A- 2333 CCUUUCUUCUUUCAUAUC 6974-6994 A- 2334 UGGGAUAUGAAAGAA 6972-6994 800849.2 1533475.1 CCA 1533476.1 GAAAGGCU AD- A- 2335 CAUCUUUUCACAGGAUUG 6573-6593 A- 2336 UUACAAUCCUGUGAAA 6571-6593 800487.1 1532751.1 UAA 1532752.1 AGAUGAC AD- A- 2337 CUGUUGGAAAUAGGUUU 8222-8242 A- 2338 UUCAAAACCUAUUUCC 8220-8242 801835.1 1535447.1 UGAA 1535448.1 AACAGGC AD- A- 2339 GGGAGAUGGAUUCUCUUC 5859-5879 A- 2340 UACGAAGAGAAUCCAU 5857-5879 799936.1 1531651.1 GUA 1531652.1 CUCCCCA AD- A- 2341 UUGAAUUCAAUCUACCGU 8328-8348 A- 2342 UUAACGGUAGAUUGA 8326-8348 801884.2 1535545.1 UAA 1535546.1 AUUCAAAU AD- A- 2343 UCAUCUUAGGCUAUUUGA 8122-8142 A- 2344 UGUUCAAAUAGCCUAA 8120-8142 801747.2 1535271.1 ACA 1535272.1 GAUGAAU AD- A- 2345 UGAUUCUUUAAGAAUCGU 6449-6469 A- 2346 UUUACGAUUCUUAAA 6447-6469 800387.2 1532551.1 AAA 1532552.1 GAAUCAUC AD- A- 2347 GUAAUGGACAUUAGUUAU 6714-6734 A- 2348 UUCAUAACUAAUGUCC 6712-6734 800606.2 1532989.1 GAA 1532990.1 AUUACUU AD- A- 2349 UUGAGACUGACACAUUGU 9699-9719 A- 2350 UUUACAAUGUGUCAG 9697-9719 802945.2 1537660.1 AAA 1537661.1 UCUCAAGU AD- A- 2351 GAAUUCAAUCUACCGUUA 8330-8350 A- 2352 UAAUAACGGUAGAUU 8328-8350 801886.2 1535549.1 UUA 1535550.1 GAAUUCAA AD- A- 2353 AUGAUUCUUUAAGAAUCG 6448-6468 A- 2354 UUACGAUUCUUAAAGA 6446-6468 800386.2 1532549.1 UAA 1532550.1 AUCAUCA AD- A- 2355 AGCCUGUUGGAAAUAGGU 8219-8239 A- 2356 UAAACCUAUUUCCAAC 8217-8239 801832.1 1535441.1 UUA 1535442.1 AGGCUUG AD- A- 2357 CGUGCUUAUAGACGUUAC 5990-6010 A- 2358 UCGGUAACGUCUAUAA 5988-6010 800060.1 1531899.1 CGA 1531900.1 GCACGCU AD- A- 2359 UUUAGUGGCAAACACUCU 4114-4134 A- 2360 UCAAGAGUGUUUGCCA 4112-4134 798332.1 1528540.1 UGA 1528541.1 CUAAAGU AD- A- 2361 ACCUCUCUUUCCAUGUAG 8705-8725 A- 2362 UAUCUACAUGGAAAGA 8703-8725 802141.2 1536059.1 AUA 1536060.1 GAGGUCU AD- A- 2363 CAACUUACUUUCCUAAAU 7456-7476 A- 2364 UUAAUUUAGGAAAGU 7454-7476 801251.1 1534279.1 UAA 1534280.1 AAGUUGGU AD- A- 2365 GCUGAACCUAUGAAUUCC 3725-3745 A- 2366 UUCGGAAUUCAUAGG 3723-3745 797963.1 1527829.1 GAA 1527830.1 UUCAGCCU AD- A- 2367 UAUCAAAAUAUUCUCGAA 6359-6379 A- 2368 UCCUUCGAGAAUAUU 6357-6379 800297.2 1532371.1 GGA 1532372.1 UUGAUAAA AD- A- 2369 ACAUCCGUUAUUACUUUG 8033-8053 A- 2370 UCUCAAAGUAAUAACG 8031-8053 801658.2 1535093.1 AGA 1535094.1 GAUGUUG AD- A- 2371 AGACAUUUGUCCUAAUCU 8051-8071 A- 2372 UGUAGAUUAGGACAA 8049-8071 801676.2 1535129.1 ACA 1535130.1 AUGUCUCA AD- A- 2373 UGCCACUGAAGAAAGUAC 5593-5613 A- 2374 UCAGUACUUUCUUCAG 5591-5613 799683.1 1531160.1 UGA 1531161.1 UGGCAAC AD- A- 2375 UCAUCUUUUCACAGGAUU 6572-6592 A- 2376 UACAAUCCUGUGAAAA 6570-6592 800486.1 1532749.1 GUA 1532750.1 GAUGACA AD- A- 2377 CGGACUUGGUUACCUAUC 4474-4494 A- 2378 UGAGAUAGGUAACCAA 4472-4494 798672.1 1529207.1 UCA 1529208.1 GUCCGAC AD- A- 2379 CUCUUUCCAUGUAGAUUA 8709-8729 A- 2380 UAGUAAUCUACAUGGA 8707-8729 802145.2 1536067.1 CUA 1536068.1 AAGAGAG AD- A- 2381 ACAACUUUCACUAAUUUG 7834-7854 A- 2382 UAGCAAAUUAGUGAAA 7832-7854 801540.2 1534857.1 CUA 1534858.1 GUUGUUU AD- A- 2383 UACAACAUCCGUUAUUAC 8029-8049 A- 2384 UAAGUAAUAACGGAU 8027-8049 801654.2 1535085.1 UUA 1535086.1 GUUGUAAA AD- A- 2385 AAUGUCGGACUUGGUUAC 4469-4489 A- 2386 UAGGUAACCAAGUCCG 4467-4489 798667.1 1529197.1 CUA 1529198.1 ACAUUAU AD- A- 2387 ACAACAUCCGUUAUUACU 8030-8050 A- 2388 UAAAGUAAUAACGGAU 8028-8050 801655.2 1535087.1 UUA 1535088.1 GUUGUAA AD- A- 2389 CUUCUUAGCCUUGUUUAG 1348-1368 A- 2390 UGCCUAAACAAGGCUA 1346-1368 795826.1 1523683.1 GCA 1523684.1 AGAAGGC AD- A- 2391 ACACAGGUAGAAUGUAGU 7770-7790 A- 2392 UAAACUACAUUCUACC 7768-7790 801490.2 1534757.1 UUA 1534758.1 UGUGUAG AD- A- 2393 CUGAACCUAUGAAUUCCG 3726-3746 A- 2394 UAUCGGAAUUCAUAG 3724-3746 797964.1 1527831.1 AUA 1527832.1 GUUCAGCC AD- A- 2395 AUUCUUUAAGAAUCGUAA 6451-6471 A- 2396 UUCUUACGAUUCUUA 6449-6471 800389.2 1532555.1 GAA 1532556.1 AAGAAUCA AD- A- 2397 GAUUCUUUAAGAAUCGUA 6450-6470 A- 2398 UCUUACGAUUCUUAAA 6448-6470 800388.2 1532553.1 AGA 1532554.1 GAAUCAU AD- A- 2399 GUUUCAGGAAUGUCUACU 8614-8634 A- 2400 UCAAGUAGACAUUCCU 8612-8634 802070.2 1535917.1 UGA 1535918.1 GAAACAA AD- A- 2401 UAUAGAAACAAAGAUUUA 7958-7978 A- 2402 UCAUAAAUCUUUGUU 7956-7978 801601.2 1534979.1 UGA 1534980.1 UCUAUAGG AD- A- 2403 UUACAACAUCCGUUAUUA 8028-8048 A- 2404 UAGUAAUAACGGAUG 8026-8048 801653.1 1535083.1 CUA 1535084.1 UUGUAAAA AD- A- 2405 UUUCAGGAAUGUCUACUU 8615-8635 A- 2406 UACAAGUAGACAUUCC 8613-8635 802071.2 1535919.1 GUA 1535920.1 UGAAACA AD- A- 2407 GAUAAUAGUCUCUUAAAC 7153-7173 A- 2408 UGAGUUUAAGAGACU 7151-7173 800968.2 1533713.1 UCA 1533714.1 AUUAUCAG AD- A- 2409 AGAGGUUAUUCUAUAUUU 6784-6804 A- 2410 UCAAAAUAUAGAAUAA 6782-6804 800667.2 1533111.1 UGA 1533112.1 CCUCUGU AD- A- 2411 UCACAACCACACUAAAACG 5937-5957 A- 2412 UCCGUUUUAGUGUGG 5935-5957 800008.2 1531795.1 GA 1531796.1 UUGUGAUG AD- A- 2413 ACACAAUUUCUUCUUAGC 8500-8520 A- 2414 UAUGCUAAGAAGAAAU 8498-8520 802016.2 1535809.1 AUA 1535810.1 UGUGUUG AD- A- 2415 UCAUCCUGGAAGUUCAGU 5458-5478 A- 2416 UCAACUGAACUUCCAG 5456-5478 799549.1 1530912.1 UGA 1530913.1 GAUGAAC AD- A- 2417 UUGCAUCAGAACCAAUUU 6826-6846 A- 2418 UAUAAAUUGGUUCUG 6824-6846 800706.2 1533189.1 AUA 1533190.1 AUGCAAUG AD- A- 2419 UUCAUCUUAGGCUAUUUG 8121-8141 A- 2420 UUUCAAAUAGCCUAAG 8119-8141 801746.2 1535269.1 AAA 1535270.1 AUGAAUG AD- A- 2421 GAUUCUUUAUACCAUCUU 8096-8116 A- 2422 UCUAAGAUGGUAUAA 8094-8116 801721.2 1535219.1 AGA 1535220.1 AGAAUCUU AD- A- 2423 AUAAUCGCUGAACUUAUU 8786-8806 A- 2424 UGUAAUAAGUUCAGC 8784-8806 802205.2 1536187.1 ACA 1536188.1 GAUUAUAA AD- A- 2425 AUUUGUCCUAAUCUACGU 8055-8075 A- 2426 UAUACGUAGAUUAGG 8053-8075 801680.2 1535137.1 AUA 1535138.1 ACAAAUGU AD- A- 2427 UUUUACAUCUGCCUUGUC 6556-6576 A- 2428 UAUGACAAGGCAGAUG 6554-6576 800470.1 1532717.1 AUA 1532718.1 UAAAAGU AD- A- 2429 ACAUUUGUCCUAAUCUAC 8053-8073 A- 2430 UACGUAGAUUAGGACA 8051-8073 801678.2 1535133.1 GUA 1535134.1 AAUGUCU AD- A- 2431 UGUUUAGUCAUCCUUUCG 7207-7227 A- 2432 UAGCGAAAGGAUGACU 7205-7227 801022.2 1533821.1 CUA 1533822.1 AAACAAU AD- A- 2433 UCUCCUUAAAAUUCUAUG 7532-7552 A- 2434 UAUCAUAGAAUUUUA 7530-7552 801309.2 1534395.1 AUA 1534396.1 AGGAGAAG AD- A- 2435 ACAGGAUUGUAAUUAGUC 6582-6602 A- 2436 UAAGACUAAUUACAAU 6580-6602 800496.2 1532769.1 UUA 1532770.1 CCUGUGA AD- A- 2437 UAGGUUCAUUCAUCUUAG 8113-8133 A- 2438 UGCCUAAGAUGAAUGA 8111-8133 801738.2 1535253.1 GCA 1535254.1 ACCUAAG AD- A- 2439 AACAACUUUCACUAAUUU 7833-7853 A- 2440 UGCAAAUUAGUGAAA 7831-7853 801539.2 1534855.1 GCA 1534856.1 GUUGUUUU AD- A- 2441 AAGCCUUUGAUAUUAGUA 4842-4862 A- 2442 UGAUACUAAUAUCAAA 4840-4862 799010.2 1529846.1 UCA 1529847.1 GGCUUGA AD- A- 2443 CUUUCUUCUUUCAUAUCC 6975-6995 A- 2444 UAGGGAUAUGAAAGA 6973-6995 800850.2 1533477.1 CUA 1533478.1 AGAAAGGC AD- A- 2445 UCACAGGAUUGUAAUUAG 6580-6600 A- 2446 UGACUAAUUACAAUCC 6578-6600 800494.2 1532765.1 UCA 1532766.1 UGUGAAA AD- A- 2447 UUGCCCUUAUGAAUGUUA 4410-4430 A- 2448 UACUAACAUUCAUAAG 4408-4430 798614.1 1529091.1 GUA 1529092.1 GGCAAAA AD- A- 2449 CAUCAGAACCAAUUUAUA 6829-6849 A- 2450 UCAUAUAAAUUGGUU 6827-6849 800709.2 1533195.1 UGA 1533196.1 CUGAUGCA AD- A- 2451 AUUCAAUCUACCGUUAUU 8332-8352 A- 2452 UGAAAUAACGGUAGA 8330-8352 801888.2 1535553.1 UCA 1535554.1 UUGAAUUC AD- A- 2453 UUUCGCUGUAAGCAAAGU 7220-7240 A- 2454 UCAACUUUGCUUACAG 7218-7240 801035.2 1533847.1 UGA 1533848.1 CGAAAGG AD- A- 2455 AUUGUUUAGUCAUCCUUU 7205-7225 A- 2456 UCGAAAGGAUGACUAA 7203-7225 801020.2 1533817.1 CGA 1533818.1 ACAAUAC AD- A- 2457 GAGACAUUUGUCCUAAUC 8050-8070 A- 2458 UUAGAUUAGGACAAA 8048-8070 801675.2 1535127.1 UAA 1535128.1 UGUCUCAA AD- A- 2459 UUGCCAACUUGCUCUCUU 7433-7453 A- 2460 UGCAAGAGAGCAAGUU 7431-7453 801228.2 1534233.1 GCA 1534234.1 GGCAAGA AD- A- 2461 AUGUAUAUUUGACCUAGU 4816-4836 A- 2462 UUCACUAGGUCAAAUA 4814-4836 798984.1 1529794.1 GAA 1529795.1 UACAUCC AD- A- 2463 CACAGGAUUGUAAUUAGU 6581-6601 A- 2464 UAGACUAAUUACAAUC 6579-6601 800495.2 1532767.1 CUA 1532768.1 CUGUGAA AD- A- 2465 GAUGUUUGACAGGUUCGU 8404-8424 A- 2466 UACACGAACCUGUCAA 8402-8424 801957.2 1535691.1 GUA 1535692.1 ACAUCUU AD- A- 2467 UAGCUGUAGACAUCUAGU 7625-7645 A- 2468 UAAACUAGAUGUCUAC 7623-7645 801399.2 1534575.1 UUA 1534576.1 AGCUAAU AD- A- 2469 UACACAGGUAGAAUGUAG 7769-7789 A- 2470 UAACUACAUUCUACCU 7767-7789 801489.2 1534755.1 UUA 1534756.1 GUGUAGC AD- A- 2471 AGUCUCUUAAACUCUUUU 7159-7179 A- 2472 UACAAAAGAGUUUAAG 7157-7179 800974.2 1533725.1 GUA 1533726.1 AGACUAU AD- A- 2473 AUCACAACCACACUAAAAC 5936-5956 A- 2474 UCGUUUUAGUGUGGU 5934-5956 800007.2 1531793.1 GA 1531794.1 UGUGAUGG AD- A- 2475 CAUUUGUCCUAAUCUACG 8054-8074 A- 2476 UUACGUAGAUUAGGA 8052-8074 801679.2 1535135.1 UAA 1535136.1 CAAAUGUC AD- A- 2477 CUGCCAAGUUAACAUAGA 3793-3813 A- 2478 UACUCUAUGUUAACU 3791-3813 798031.1 1527964.1 GUA 1527965.1 UGGCAGCA AD- A- 2479 AUUAGCUGUAGACAUCUA 7623-7643 A- 2480 UACUAGAUGUCUACAG 7621-7643 801397.2 1534571.1 GUA 1534572.1 CUAAUGC AD- A- 2481 GUCUCUUAAACUCUUUUG 7160-7180 A- 2482 UGACAAAAGAGUUUAA 7158-7180 800975.2 1533727.1 UCA 1533728.1 GAGACUA AD- A- 2483 GACAUUUGUCCUAAUCUA 8052-8072 A- 2484 UCGUAGAUUAGGACAA 8050-8072 801677.2 1535131.1 CGA 1535132.1 AUGUCUC AD- A- 2485 UUCUUUAUACCAUCUUAG 8098-8118 A- 2486 UACCUAAGAUGGUAUA 8096-8118 801723.2 1535223.1 GUA 1535224.1 AAGAAUC AD- A- 2487 CACAGGUAGAAUGUAGUU 7771-7791 A- 2488 UAAAACUACAUUCUAC 7769-7791 801491.2 1534759.1 UUA 1534760.1 CUGUGUA AD- A- 2489 AUGUAGAUUACUGUUUG 8717-8737 A- 2490 UGUACAAACAGUAAUC 8715-8737 802153.2 1536083.1 UACA 1536084.1 UACAUGG AD- A- 2491 UCACUUGUAUACAAUCCC 7325-7345 A- 2492 UACGGGAUUGUAUAC 7323-7345 801140.2 1534057.1 GUA 1534058.1 AAGUGAUG AD- A- 2493 AUUCAUCUUAGGCUAUUU 8120-8140 A- 2494 UUCAAAUAGCCUAAGA 8118-8140 801745.2 1535267.1 GAA 1535268.1 UGAAUGA AD- A- 2495 CAUUCAUCUUAGGCUAUU 8119-8139 A- 2496 UCAAAUAGCCUAAGAU 8117-8139 801744.2 1535265.1 UGA 1535266.1 GAAUGAA AD- A- 2497 AGAGCUUAUUAAGUAUAA 8670-8690 A- 2498 UGCUUAUACUUAAUA 8668-8690 802106.2 1535989.1 GCA 1535990.1 AGCUCUUU AD- A- 2499 UGAUGAUUCUUUAAGAAU 6446-6466 A- 2500 UCGAUUCUUAAAGAAU 6444-6466 800384.2 1532545.1 CGA 1532546.1 CAUCAGU AD- A- 2501 CAACAGAUGUUAGACCGU 1568-1588 A- 2502 UAGACGGUCUAACAUC 1566-1588 796041.1 1524103.1 CUA 1524104.1 UGUUGAA

TABLE 5A Exemplary Human SCN9A siRNA Modified Single Strands and Duplex Sequences Column 1 indicates duplex name and the number following the decimal point in a duplex name merely refers to a batch production number. Column  2 indicates the name of the sense sequence. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the modified sequence of a sense strand suitable for use in a duplex described herein. Column 5 indicates the antisense sequence name. Column 6 indicates the sequence ID for the sequence of column 7. Column 7 provides the sequence of a modified antisense strand suitable for use in a duplex described herein, e.g., a duplex comprising the sense sequence in the same row of the table. Column 8 indicates the position in the target mRNA (NM_002977.3) that is complementary to the antisense strand of Column 7. Column 9 indicated the sequence ID for the sequence of column 8. SEQ ID Seq ID NO: Sense Seq ID Antisense NO: mRNA target (mRNA Duplex sequence NO: Sense sequence sequence (anti Antisense sequence sequence target) Name name (sense) (5′-3′) name sense) (5′-3′) in NM_002977.3 AD- A- 2503 ususgug(Ahd)cudTu A- 2593 VPusdCsacdTadAacuu UAUUGUGACUUUAAG 3516 961208.1 1812652.1 dAaguuuagugaL96 1812653.1 dAadAgdTcacaasusa UUUAGUGG AD- A- 2504 usasuug(Uhd)gadCu A- 2594 VPusdCsuadAadCuua CUUAUUGUGACUUUA 3517 961207.1 1812650.1 dTuaaguuuagaL96 1812651.1 adAgdTcdAcaauasasg AGUUUAGU AD- A- 2505 ususcug(Uhd)gudAg A- 2595 VPusdGsugdAadTucu GCUUCUGUGUAGGAG 3518 1010662.1 1851786.1 dGagaauucacaL96 1875200.1 cdCudAcdAcagaasgsc AAUUCACU AD- A- 2506 csasuga(Uhd)cudTc A- 2596 VPusdCsuadCgdAcaa UACAUGAUCUUCUUU 3519 961188.1 1812612.1 dTuugucguagaL96 1812613.1 adGadAgdAucaugsus GUCGUAGU a AD- A- 2507 usgsuag(Ghd)agdAa A- 2597 VPusdGsaadAadGuga UGUGUAGGAGAAUUC 3520 1010663.1 1851796.1 dTucacuuuucaL96 1875201.1 adTudCudCcuacascsa ACUUUUCU AD- A- 2508 usgsucg(Ahd)gudAc A- 2598 VPusdCsagdTadAaag AAUGUCGAGUACACUU 3521 1010661.1 1851664.1 dAcuuuuacugaL96 1875199.1 udGudAcdTcgacasus UUACUGG u AD- A- 2509 asusgau(Chd)uudCu A- 2599 VPusdAscudAcdGaca ACAUGAUCUUCUUUG 3522 961189.1 1812614.1 dTugucguaguaL96 1812615.1 adAgdAadGaucausgs UCGUAGUG u AD- A- 2510 asuscug(Ahd)gadCu A- 2600 VPusdCsggdCadAauu GGAUCUGAGACUGAA 3523 1010671.1 1853827.1 dGaauuugccgaL96 1875209.1 cdAgdTcdTcagauscsc UUUGCCGA AD- A- 2511 usgsauc(Uhd)ucdTu A- 2601 VPusdCsacdTadCgaca CAUGAUCUUCUUUGU 3524 961190.1 1812616.1 dTgucguagugaL96 1812617.1 dAadGadAgaucasusg CGUAGUGA AD- A- 2512 asasggg(Ahd)aadAc A- 2602 VPusdAscgdGadAgau CAAAGGGAAAACAAUC 3525 961179.1 1812594.1 dAaucuuccguaL96 1812595.1 udGudTudTcccuusus UUCCGUU g AD- A- 2513 asgscuu(Ghd)aadGu A- 2603 VPusdGsucdTadAuuu UAAGCUUGAAGUAAAA 3526 961342.1 1812920.1 dAaaauuagacaL96 1812921.1 udAcdTudCaagcususa UUAGACC AD- A- 2514 usgscua(Uhd)agdGa A- 2604 VPusdAsgadCcdAaau CUUGCUAUAGGAAAU 3527 1010673.1 1854804.1 dAauuuggucuaL96 1875211.1 udTcdCudAuagcasasg UUGGUCUU AD- A- 2515 asuscuu(Chd)uudTg A- 2605 VPusdAsucdAcdTacga UGAUCUUCUUUGUCG 3528 961192.1 1812620.1 dTcguagugauaL96 1812621.1 dCadAadGaagauscsa UAGUGAUU AD- A- 2516 gsasucu(Uhd)cudTu A- 2606 VPusdTscadCudAcgac AUGAUCUUCUUUGUC 3529 961191.1 1812618.1 dGucguagugaaL96 1812619.1 dAadAgdAagaucsasu GUAGUGAU AD- A- 2517 ususauu(Ghd)cadTc A- 2607 VPusdGsuadTadCaag AUUUAUUGCAUCACU 3530 1010693.1 1863139.1 dAcuuguauacaL96 1875231.1 udGadTgdCaauaasas UGUAUACA u AD- A- 2518 csasaca(Chd)aadTu A- 2608 VPusdGscudAadGaag AACAACACAAUUUCUU 3531 961334.1 1812904.1 dTcuucuuagcaL96 1812905.1 adAadTudGuguugsus CUUAGCA u AD- A- 2519 csusguu(Ghd)gadAa A- 2609 VPusdTscadAadAccua GCCUGUUGGAAAUAG 3532 1010697.1 1864516.1 dTagguuuugaaL96 1875235.1 dTudTcdCaacagsgsc GUUUUGAU AD- A- 2520 ususugu(Ahd)gadTc A- 2610 VPusdGsuadAudTgca CUUUUGUAGAUCUUG 3533 961203.1 1812642.1 dTugcaauuacaL96 1812643.1 adGadTcdTacaaasasg CAAUUACC AD- A- 2521 usgsguu(Uhd)cadGc A- 2611 VPusdCsugdAadTcug UGUGGUUUCAGCACA 3534 1010664.1 1852529.1 dAcagauucagaL96 1875202.1 udGcdTgdAaaccascsa GAUUCAGG AD- A- 2522 ususgau(Ahd)gudTa A- 2612 VPusdGscadAadCuag UUUUGAUAGUUACCU 3535 1010698.1 1865925.1 dCcuaguuugcaL96 1875236.1 gdTadAcdTaucaasasa AGUUUGCA AD- A- 2523 ascsaug(Ahd)ucdTu A- 2613 VPusdTsacdGadCaaag CUACAUGAUCUUCUUU 3536 961187.1 1812610.1 dCuuugucguaaL96 1812611.1 dAadGadTeaugusasg GUCGUAG AD- A- 2524 gsusuug(Ahd)acdAc A- 2614 VPusdCsgadAadGauu AGGUUUGAACACAAAU 3537 961350.1 1812936.1 dAaaucuuucgaL96 1812937.1 udGudGudTcaaacscs CUUUCGG u AD- A- 2525 usgsaga(Chd)ugdAc A- 2615 VPusdAsuudAcdAaug CUUGAGACUGACACAU 3538 1010700.1 1866708.1 dAcauuguaauaL96 1875238.1 udGudCadGucucasas UGUAAUA g AD- A- 2526 asusguc(Ghd)agdTa A- 2616 VPusdAsgudAadAagu AAAUGUCGAGUACACU 3539 961182.1 1812600.1 dCacuuuuacuaL96 1812601.1 gdTadCudCgacaususu UUUACUG AD- A- 2527 usgsaua(Ghd)uudAc A- 2617 VPusdTsgcdAadAcuag UUUGAUAGUUACCUA 3540 1010699.1 1865927.1 dCuaguuugcaaL96 1875237.1 dGudAadCuaucasasa GUUUGCAA AD- A- 2528 usasuau(Uhd)uudA A- 2618 VPusdAsacdGgdAugu GAUAUAUUUUACAACA 3541 1010696.1 1864159.1 cdAacauccguuaL96 1875234.1 udGudAadAauauasus UCCGUUA c AD- A- 2529 csusuua(Uhd)acdCa A- 2619 VPusdGsaadCcdTaaga UUCUUUAUACCAUCUU 3542 961321.1 1812878.1 dTcuuagguucaL96 1812879.1 dTgdGudAuaaagsasa AGGUUCA AD- A- 2530 asusgua(Chd)agdAg A- 2620 VPusdAsuadGadAuaa CAAUGUACAGAGGUUA 3543 961279.1 1812794.1 dGuuauucuauaL96 1812795.1 cdCudCudGuacausus UUCUAUA g AD- A- 2531 gscsguu(Ghd)uadG A- 2621 VPusdGsgadGadTagg AGGCGUUGUAGUUCC 3544 1010672.1 1854206.1 udTccuaucuccaL96 1875210.1 adAcdTadCaacgcscsu UAUCUCCU AD- A- 2532 asasguc(Ahd)agdTu A- 2622 VPusdAsacdGadTuug GCAAGUCAAGUUCCAA 3545 961226.1 1812688.1 dCcaaaucguuaL96 1812689.1 gdAadCudTgacuusgsc AUCGUUC AD- A- 2533 gscsaag(Uhd)cadAg A- 2623 VPusdCsgadTudTggaa CUGCAAGUCAAGUUCC 3546 961225.1 1812686.1 dTuccaaaucgaL96 1812687.1 dCudTgdAcuugcsasg AAAUCGU AD- A- 2534 ususggc(Ahd)gadAa A- 2624 VPusdAsuadAudCagg AAUUGGCAGAAACCCU 3547 1010665.1 1852599.1 dCccugauuauaL96 1875203.1 gdTudTcdTgccaasusu GAUUAUG AD- A- 2535 csusgau(Uhd)ucdCu A- 2625 VPusdCsacdCudTucu CUCUGAUUUCCUAAGA 3548 961259.1 1812754.1 dAagaaaggugaL96 1812755.1 udAgdGadAaucagsas AAGGUGG g AD- A- 2536 uscsgug(Ghd)cudCc A- 2626 VPusdCsagdAadAaca AUUCGUGGCUCCUUG 3549 961201.1 1812638.1 dTuguuuucugaL96 1812639.1 adGgdAgdCcacgasasu UUUUCUGC AD- A- 2537 gsuscuu(Uhd)acdTg A- 2627 VPusdGscadAadGauu UGGUCUUUACUGGAA 3550 1010674.1 1854836.1 dGaaucuuugcaL96 1875212.1 cdCadGudAaagacscsa UCUUUGCA AD- A- 2538 csusucu(Ghd)aadAc A- 2628 VPusdCsagdTudTggau UUCUUCUGAAACAUCC 3551 1010670.1 1853318.1 dAuccaaacugaL96 1875208.1 dGudTudCagaagsasa AAACUGA AD- A- 2539 ususgcu(Ahd)uadGg A- 2629 VPusdGsacdCadAauu ACUUGCUAUAGGAAAU 3552 961206.1 1812648.1 dAaauuuggucaL96 1812649.1 udCcdTadTagcaasgsu UUGGUCU AD- A- 2540 asgsccu(Ghd)uudGg A- 2630 VPusdAsaadCcdTauu CAAGCCUGUUGGAAAU 3553 961326.1 1812888.1 dAaauagguuuaL96 1812889.1 udCcdAadCaggcususg AGGUUUU AD- A- 2541 asusguu(Uhd)cudAg A- 2631 VPusdAsucdAadAuca GUAUGUUUCUAGCUG 3554 961239.1 1812714.1 dCugauuugauaL96 1812715.1 gdCudAgdAaacausasc AUUUGAUU AD- A- 2542 ususgca(Ahd)gcdCu A- 2632 VPusdCsucdAcdAuaa GGUUGCAAGCCUCUUA 3555 1010660.1 1850886.1 dCuuaugugagaL96 1875198.1 gdAgdGcdTugcaascsc UGUGAGG AD- A- 2543 ususuag(Uhd)ggdCa A- 2633 VPusdCsaadGadGugu ACUUUAGUGGCAAACA 3556 1010677.1 1857611.1 dAacacucuugaL96 1875215.1 udTgdCcdAcuaaasgsu CUCUUGG AD- A- 2544 gsascuu(Ahd)ccdTu A- 2634 VPusdCsaadTadCucua AAGACUUACCUUUAGA 3557 1010690.1 1862528.1 dTagaguauugaL96 1875228.1 dAadGgdTaagucsusu GUAUUGU AD- A- 2545 gsgscgu(Uhd)gudAg A- 2635 VPusdGsagdAudAgga AAGGCGUUGUAGUUC 3558 961202.1 1812640.1 dTuccuaucucaL96 1812641.1 adCudAcdAacgccsusu CUAUCUCC AD- A- 2546 ususugu(Chd)gudAg A- 2636 VPusdAsggdAadAauc UCUUUGUCGUAGUGA 3559 1010668.1 1852884.1 dTgauuuuccuaL96 1875206.1 adCudAcdGacaaasgsa UUUUCCUG AD- A- 2547 csasacu(Uhd)acdTu A- 2637 VPusdTsaadTudTagga ACCAACUUACUUUCCU 3560 1010694.1 1863376.1 dTccuaaauuaaL96 1875232.1 dAadGudAaguugsgsu AAAUUAU AD- A- 2548 gsusaug(Uhd)uudC A- 2638 VPusdCsaadAudCagc AGGUAUGUUUCUAGC 3561 1010679.1 1859377.1 udAgcugauuugaL96 1875217.1 udAgdAadAcauacscs UGAUUUGA U AD- A- 2549 gsascag(Ahd)gadTg A- 2639 VPusdAsgudAadAuca GAGACAGAGAUGAUG 3562 961257.1 1812750.1 dAugauuuacuaL96 1812751.1 udCadTcdTcugucsusc AUUUACUC AD- A- 2550 gsasgau(Ghd)gadTu A- 2640 VPusdGsaadCgdAaga GGGAGAUGGAUUCUC 3563 961245.1 1812726.1 dCucuucguucaL96 1812727.1 gdAadTcdCaucucscsc UUCGUUCA AD- A- 2551 ususccu(Ghd)audAu A- 2641 VPusdAsacdTadAcugc CCUUCCUGAUAUGCAG 3564 1010692.1 1863006.1 dGcaguuaguuaL96 1875230.1 dAudAudCaggaasgsg UUAGUUG AD- A- 2552 csasccu(Uhd)cudCc A- 2642 VPusdAsgadAudTuua GUCACCUUCUCCUUAA 3565 1010695.1 1863481.1 dTuaaaauucuaL96 1875233.1 adGgdAgdAaggugsas AAUUCUA c AD- A- 2553 csusgau(Ahd)audAg A- 2643 VPusdGsuudTadAgag UACUGAUAAUAGUCUC 3566 961285.1 1812806.1 dTcucuuaaacaL96 1812807.1 adCudAudTaucagsus UUAAACU a AD- A- 2554 csusaaa(Uhd)uadTg A- 2644 VPusdAsgadTudAcuu UCCUAAAUUAUGGAAG 3567 961300.1 1812836.1 dGaaguaaucuaL96 1812837.1 cdCadTadAuuuagsgsa UAAUCUU AD- A- 2555 uscsuuu(Ahd)uadCc A- 2645 VPusdAsacdCudAaga AUUCUUUAUACCAUCU 3568 961320.1 1812876.1 dAucuuagguuaL96 1812877.1 udGgdTadTaaagasasu UAGGUUC AD- A- 2556 gsgsaga(Uhd)ggdAu A- 2646 VPusdAsacdGadAgag GGGGAGAUGGAUUCU 3569 1010684.1 1860794.1 dTcucuucguuaL96 1875222.1 adAudCcdAucuccscsc CUUCGUUC AD- A- 2557 usgsaau(Ahd)uadCa A- 2647 VPusdTsccdTadAuacu GCUGAAUAUACAAGUA 3570 1010669.1 1853216.1 dAguauuaggaaL96 1875207.1 dTgdTadTauucasgsc UUAGGAG AD- A- 2558 gsusuuc(Uhd)agdCu A- 2648 VPusdCsaadTcdAaauc AUGUUUCUAGCUGAU 3571 1010680.1 1859383.1 dGauuugauugaL96 1875218.1 dAgdCudAgaaacsasu UUGAUUGA AD- A- 2559 asgsuca(Ahd)gudTc A- 2649 VPusdGsaadCgdAuuu CAAGUCAAGUUCCAAA 3572 961227.1 1812690.1 dCaaaucguucaL96 1812691.1 gdGadAcdTugacusus UCGUUCC g AD- A- 2560 csasucu(Ghd)uudGg A- 2650 VPusdGsuadGadAuau CCCAUCUGUUGGAAUA 3573 961243.1 1812722.1 dAauauucuacaL96 1812723.1 udCcdAadCagaugsgsg UUCUACU AD- A- 2561 csusgaa(Chd)cudAu A- 2651 VPusdAsucdGgdAauu GGCUGAACCUAUGAAU 3574 961221.1 1812678.1 dGaauuccgauaL96 1812679.1 cdAudAgdGuucagscsc UCCGAUG AD- A- 2562 csusuuu(Chd)acdAg A- 2652 VPusdAsaudTadCaau AUCUUUUCACAGGAU 3575 961271.1 1812778.1 dGauuguaauuaL96 1812779.1 cdCudGudGaaaagsas UGUAAUUA U AD- A- 2563 asgscgu(Ghd)cudTa A- 2653 VPusdGsuadAcdGucu UCAGCGUGCUUAUAGA 3576 961251.1 1812738.1 dTagacguuacaL96 1812739.1 adTadAgdCacgcusgsa CGUUACC AD- A- 2564 csusucc(Uhd)gadTa A- 2654 VPusdAscudAadCugc UCCUUCCUGAUAUGCA 3577 961296.1 1812828.1 dTgcaguuaguaL96 1812829.1 adTadTcdAggaagsgsa GUUAGUU AD- A- 2565 gsgsaag(Ahd)aadGg A- 2655 VPusdCsagdAcdAuga AUGGAAGAAAGGUUC 3578 961246.1 1812728.1 dTucaugucugaL96 1812729.1 adCcdTudTcuuccsasu AUGUCUGC AD- A- 2566 gsusaga(Ahd)aadCu A- 2656 VPusdCsagdAudGuaa AUGUAGAAAACUUUU 3579 1010688.1 1861826.1 dTuuacaucugaL96 1875226.1 adAgdTudTucuacsasu ACAUCUGC AD- A- 2567 uscsauc(Uhd)uudTc A- 2657 VPusdAscadAudCcug UGUCAUCUUUUCACAG 3580 961269.1 1812774.1 dAcaggauuguaL96 1812775.1 udGadAadAgaugascs GAUUGUA a AD- A- 2568 gscscca(Ahd)aadTa A- 2658 VPusdCsuadTudAuca CUGCCCAAAAUACUGA 3581 1010691.1 1862804.1 dCugauaauagaL96 1875229.1 gdTadTudTugggcsasg UAAUAGU AD- A- 2569 ususuua(Chd)audCu A- 2659 VPusdAsugdAcdAagg ACUUUUACAUCUGCCU 3582 1010689.1 1861844.1 dGccuugucauaL96 1875227.1 cdAgdAudGuaaaasgs UGUCAUC u AD- A- 2570 usasggc(Uhd)aadTg A- 2660 VPusdAsaudCudTggg UUUAGGCUAAUGACCC 3583 1010667.1 1852732.1 dAcccaagauuaL96 1875205.1 udCadTudAgccuasasa AAGAUUA AD- A- 2571 csgsugc(Uhd)uadTa A- 2661 VPusdCsggdTadAcguc AGCGUGCUUAUAGACG 3584 961252.1 1812740.1 dGacguuaccgaL96 1812741.1 dTadTadAgcacgscsu UUACCGC AD- A- 2572 csusucu(Uhd)agdCc A- 2662 VPusdGsccdTadAacaa GCCUUCUUAGCCUUGU 3585 1010666.1 1852704.1 dTuguuuaggcaL96 1875204.1 dGgdCudAagaagsgsc UUAGGCU AD- A- 2573 usgsgaa(Uhd)audTc A- 2663 VPusdTsaadCadAagu GUUGGAAUAUUCUAC 3586 1010682.1 1860117.1 dTacuuuguuaaL96 1875220.1 adGadAudAuuccasas UUUGUUAG c AD- A- 2574 csusgaa(Uhd)audAc A- 2664 VPusdCscudAadTacu GGCUGAAUAUACAAGU 3587 961196.1 1812628.1 dAaguauuaggaL96 1812629.1 udGudAudAuucagscs AUUAGGA c AD- A- 2575 csusgcc(Ahd)agdTu A- 2665 VPusdAscudCudAugu UGCUGCCAAGUUAACA 3588 1010676.1 1857011.1 dAacauagaguaL96 1875214.1 udAadCudTggcagscsa UAGAGUC AD- A- 2576 usgsgau(Uhd)cudCu A- 2666 VPusdCsugdTgdAacga GAUGGAUUCUCUUCG 3589 1010686.1 1860802.1 dTcguucacagaL96 1875224.1 dAgdAgdAauccasusc UUCACAGA AD- A- 2577 gscsaaa(Ghd)gudCa A- 2667 VPusdGsagdGadAauu GAGCAAAGGUCACAAU 3590 1010675.1 1856353.1 dCaauuuccucaL96 1875213.1 gdTgdAcdCuuugcsusc UUCCUCA AD- A- 2578 usgscca(Chd)ugdAa A- 2668 VPusdCsagdTadCuuu GUUGCCACUGAAGAAA 3591 961244.1 1812724.1 dGaaaguacugaL96 1812725.1 cdTudCadGuggcasasc GUACUGA AD- A- 2579 cscsuuc(Chd)ugdAu A- 2669 VPusdCsuadAcdTgcau AUCCUUCCUGAUAUGC 3592 961295.1 1812826.1 dAugcaguuagaL96 1812827.1 dAudCadGgaaggsasu AGUUAGU AD- A- 2580 csasucu(Uhd)uudCa A- 2670 VPusdTsacdAadTccug GUCAUCUUUUCACAGG 3593 961270.1 1812776.1 dCaggauuguaaL96 1812777.1 dTgdAadAagaugsasc AUUGUAA AD- A- 2581 gsgsgag(Ahd)ugdGa A- 2671 VPusdAscgdAadGaga UGGGGAGAUGGAUUC 3594 1010683.1 1860792.1 dTucucuucguaL96 1875221.1 adTcdCadTcucccscsa UCUUCGUU AD- A- 2582 asasugu(Chd)ggdAc A- 2672 VPusdAsggdTadAccaa AUAAUGUCGGACUUG 3595 1010678.1 1858274.1 dTugguuaccuaL96 1875216.1 dGudCcdGacauusasu GUUACCUA AD- A- 2583 uscsauc(Chd)ugdGa A- 2673 VPusdCsaadCudGaac GUUCAUCCUGGAAGU 3596 1010681.1 1860028.1 dAguucaguugaL96 1875219.1 udTcdCadGgaugasasc UCAGUUGA AD- A- 2584 asusgua(Uhd)audTu A- 2674 VPusdTscadCudAgguc GGAUGUAUAUUUGAC 3597 961233.1 1812702.1 dGaccuagugaaL96 1812703.1 dAadAudAuacauscsc CUAGUGAC AD- A- 2585 asgsuca(Chd)cadCu A- 2675 VPusdAscgdAadTgcug UCAGUCACCACUCAGC 3598 961200.1 1812636.1 dCagcauucguaL96 1812637.1 dAgdTgdGugacusgsa AUUCGUG AD- A- 2586 asuscgu(Ahd)agdAg A- 2676 VPusdCsuadCadGagu GAAUCGUAAGAGAACU 3599 961267.1 1812770.1 dAacucuguagaL96 1812771.1 udCudCudTacgaususc CUGUAGG AD- A- 2587 gscsuga(Ahd)ccdTa A- 2677 VPusdTscgdGadAuuc AGGCUGAACCUAUGAA 3600 961220.1 1812676.1 dTgaauuccgaaL96 1812677.1 adTadGgdTucagcscsu UUCCGAU AD- A- 2588 csgsgac(Uhd)ugdGu A- 2678 VPusdGsagdAudAggu GUCGGACUUGGUUAC 3601 961232.1 1812700.1 dTaccuaucucaL96 1812701.1 adAcdCadAguccgsasc CUAUCUCU AD- A- 2589 asgsaug(Ghd)audTc A- 2679 VPusdTsgadAcdGaag GGAGAUGGAUUCUCU 3602 1010685.1 1860796.1 dTcuucguucaaL96 1875223.1 adGadAudCcaucuscsc UCGUUCAC AD- A- 2590 asgsacg(Uhd)uadCc A- 2680 VPusdTsgcdCudTaagc AUAGACGUUACCGCUU 3603 1010687.1 1861054.1 dGcuuaaggcaaL96 1875225.1 dGgdTadAcgucusasu AAGGCAA AD- A- 2591 usgsuag(Ahd)ucdTu A- 2681 VPusdTsggdTadAuugc UUUGUAGAUCUUGCA 3604 961204.1 1812644.1 dGcaauuaccaaL96 1812645.1 dAadGadTcuacasasa AUUACCAU AD- A- 2592 ususgcc(Chd)uudAu A- 2682 VPusdAscudAadCauu UUUUGCCCUUAUGAA 3605 961231.1 1812698.1 dGaauguuaguaL96 1812699.1 cdAudAadGggcaasas UGUUAGUC a

TABLE 5B Exemplary Human SCN9A Unmodified Single Strands and Duplex Sequences. Column 1 indicates duplex name and the number following the decimal point in a duplex name merely refers to a batch production number. Column 2 indicates the sense sequence name. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the unmodified sequence of a sense strand suitable for use in a duplex described herein. Column 5 provides the position in the target mRNA (NM_002977.3) of the sense strand of Column 4. Column 6 indicates the antisense sequence name. Column 7 indicates the sequence ID for the sequence of column 8. Column 8 provides the sequence of an antisense strand suitable for use in a duplex described herein, without specifying chemical modifications. Column 9 indicates the position in the target mRNA (NM_002977.3) that is complementary to the antisense strand of Column 8. mRNA  mRNA target Anti Seq ID target Sense Seq ID range in sense NO: range in Duplex sequence NO: Sense sequence NM_ sequence (anti antisense sequence NM_00297 Name name (sense)  (5′-3′) 002977.3 name sense)  (5′-3′) 7.3 AD- A- 2683 UUGUGACUTUAAGUUUA 2752-2772 A- 2773 UCACTAAACUUAAAGTCAC 2750-2772 961208.1 1812652.1 GUGA 1812653.1 AAUA AD- A- 2684 UAUUGUGACUTUAAGUU 2750-2770 A- 2774 UCUAAACUUAAAGTCACAA 2748-2770 961207.1 1812650.1 UAGA 1812651.1 UAAG AD- A- 2685 UUCUGUGUAGGAGAAUU 867-887 A- 2775 UGUGAATUCUCCUACACAG 865-887 1010662.1 1851786.1 CACA 1875200.1 AAGC AD- A- 2686 CAUGAUCUTCTUUGUCGU 1475-1495 A- 2776 UCUACGACAAAGAAGAUCA 1473-1495 961188.1 1812612.1 AGA 1812613.1 UGUA AD- A- 2687 UGUAGGAGAATUCACUUU 872-892 A- 2777 UGAAAAGUGAATUCUCCUA 870-892 1010663.1 1851796.1 UCA 1875201.1 CACA AD- A- 2688 UGUCGAGUACACUUUUA 803-823 A- 2778 UCAGTAAAAGUGUACTCGA 801-823 1010661.1 1851664.1 CUGA 1875199.1 CAUU AD- A- 2689 AUGAUCUUCUTUGUCGU 1476-1496 A- 2779 UACUACGACAAAGAAGAUC 1474-1496 961189.1 1812614.1 AGUA 1812615.1 AUGU AD- A- 2690 AUCUGAGACUGAAUUUG 2036-2056 A- 2780 UCGGCAAAUUCAGTCTCAG 2034-2056 1010671.1 1853827.1 CCGA 1875209.1 AUCC AD- A- 2691 UGAUCUUCTUTGUCGUAG 1477-1497 A- 2781 UCACTACGACAAAGAAGAU 1475-1497 961190.1 1812616.1 UGA 1812617.1 CAUG AD- A- 2692 AAGGGAAAACAAUCUUCC 619-639 A- 2782 UACGGAAGAUUGUTUTCCC 617-639 961179.1 1812594.1 GUA 1812595.1 UUUG AD- A- 2693 AGCUUGAAGUAAAAUUA 8697-8717 A- 2783 UGUCTAAUUUUACTUCAAG 8695-8717 961342.1 1812920.1 GACA 1812921.1 CUUA AD- A- 2694 UGCUAUAGGAAAUUUGG 2636-2656 A- 2784 UAGACCAAAUUTCCUAUAG 2634-2656 1010673.1 1854804.1 UCUA 1875211.1 CAAG AD- A- 2695 AUCUUCUUTGTCGUAGUG 1479-1499 A- 2785 UAUCACTACGACAAAGAAG 1477-1499 961192.1 1812620.1 AUA 1812621.1 AUCA AD- A- 2696 GAUCUUCUTUGUCGUAG 1478-1498 A- 2786 UTCACUACGACAAAGAAGA 1476-1498 961191.1 1812618.1 UGAA 1812619.1 UCAU AD- A- 2697 UUAUUGCATCACUUGUAU 7327-7347 A- 2787 UGUATACAAGUGATGCAAU 7325-7347 1010693.1 1863139.1 ACA 1875231.1 AAAU AD- A- 2698 CAACACAATUTCUUCUUA 8508-8528 A- 2788 UGCUAAGAAGAAATUGUG 8506-8528 961334.1 1812904.1 GCA 1812905.1 UUGUU AD- A- 2699 CUGUUGGAAATAGGUUU 8232-8252 A- 2789 UTCAAAACCUATUTCCAACA 8230-8252 1010697.1 1864516.1 UGAA 1875235.1 GGC AD- A- 2700 UUUGUAGATCTUGCAAUU 2541-2561 A- 2790 UGUAAUTGCAAGATCTACA 2539-2561 961203.1 1812642.1 ACA 1812643.1 AAAG AD- A- 2701 UGGUUUCAGCACAGAUUC 1286-1306 A- 2791 UCUGAATCUGUGCTGAAAC 1284-1306 1010664.1 1852529.1 AGA 1875202.1 CACA AD- A- 2702 UUGAUAGUTACCUAGUU 9235-9255 A- 2792 UGCAAACUAGGTAACTAUC 9233-9255 1010698.1 1865925.1 UGCA 1875236.1 AAAA AD- A- 2703 ACAUGAUCTUCUUUGUCG 1474-1494 A- 2793 UTACGACAAAGAAGATCAU 1472-1494 961187.1 1812610.1 UAA 1812611.1 GUAG AD- A- 2704 GUUUGAACACAAAUCUU 9184-9204 A- 2794 UCGAAAGAUUUGUGUTCA 9182-9204 961350.1 1812936.1 UCGA 1812937.1 AACCU AD- A- 2705 UGAGACUGACACAUUGUA 9710-9730 A- 2795 UAUUACAAUGUGUCAGUC 9708-9730 1010700.1 1866708.1 AUA 1875238.1 UCAAG AD- A- 2706 AUGUCGAGTACACUUUUA 802-822 A- 2796 UAGUAAAAGUGTACUCGAC 800-822 961182.1 1812600.1 CUA 1812601.1 AUUU AD- A- 2707 UGAUAGUUACCUAGUUU 9236-9256 A- 2797 UTGCAAACUAGGUAACUAU 9234-9256 1010699.1 1865927.1 GCAA 1875237.1 CAAA AD- A- 2708 UAUAUUUUACAACAUCCG 8032-8052 A- 2798 UAACGGAUGUUGUAAAAU 8030-8052 1010696.1 1864159.1 UUA 1875234.1 AUAUC AD- A- 2709 CUUUAUACCATCUUAGGU 8110-8130 A- 2799 UGAACCTAAGATGGUAUAA 8108-8130 961321.1 1812878.1 UCA 1812879.1 AGAA AD- A- 2710 AUGUACAGAGGUUAUUC 6788-6808 A- 2800 UAUAGAAUAACCUCUGUA 6786-6808 961279.1 1812794.1 UAUA 1812795.1 CAUUG AD- A- 2711 GCGUUGUAGUTCCUAUCU 2312-2332 A- 2801 UGGAGATAGGAACTACAAC 2310-2332 1010672.1 1854206.1 CCA 1875210.1 GCCU AD- A- 2712 AAGUCAAGTUCCAAAUCG 4391-4411 A- 2802 UAACGATUUGGAACUTGAC 4389-4411 961226.1 1812688.1 UUA 1812689.1 UUGC AD- A- 2713 GCAAGUCAAGTUCCAAAU 4389-4409 A- 2803 UCGATUTGGAACUTGACUU 4387-4409 961225.1 1812686.1 CGA 1812687.1 GCAG AD- A- 2714 UUGGCAGAAACCCUGAUU 1339-1359 A- 2804 UAUAAUCAGGGTUTCTGCC 1337-1359 1010665.1 1852599.1 AUA 1875203.1 AAUU AD- A- 2715 CUGAUUUCCUAAGAAAGG 6406-6426 A- 2805 UCACCUTUCUUAGGAAAUC 6404-6426 961259.1 1812754.1 UGA 1812755.1 AGAG AD- A- 2716 UCGUGGCUCCTUGUUUUC 1958-1978 A- 2806 UCAGAAAACAAGGAGCCAC 1956-1978 961201.1 1812638.1 UGA 1812639.1 GAAU AD- A- 2717 GUCUUUACTGGAAUCUU 2652-2672 A- 2807 UGCAAAGAUUCCAGUAAA 2650-2672 1010674.1 1854836.1 UGCA 1875212.1 GACCA AD- A- 2718 CUUCUGAAACAUCCAAAC 1726-1746 A- 2808 UCAGTUTGGAUGUTUCAGA 1724-1746 1010670.1 1853318.1 UGA 1875208.1 AGAA AD- A- 2719 UUGCUAUAGGAAAUUUG 2635-2655 A- 2809 UGACCAAAUUUCCTATAGC 2633-2655 961206.1 1812648.1 GUCA 1812649.1 AAGU AD- A- 2720 AGCCUGUUGGAAAUAGG 8229-8249 A- 2810 UAAACCTAUUUCCAACAGG 8227-8249 961326.1 1812888.1 UUUA 1812889.1 CUUG AD- A- 2721 AUGUUUCUAGCUGAUUU 5085-5105 A- 2811 UAUCAAAUCAGCUAGAAAC 5083-5105 961239.1 1812714.1 GAUA 1812715.1 AUAC AD- A- 2722 UUGCAAGCCUCUUAUGU 286-306 A- 2812 UCUCACAUAAGAGGCTUGC 284-306 1010660.1 1850886.1 GAGA 1875198.1 AACC AD- A- 2723 UUUAGUGGCAAACACUCU 4124-4144 A- 2813 UCAAGAGUGUUTGCCACUA 4122-4144 1010677.1 1857611.1 UGA 1875215.1 AAGU AD- A- 2724 GACUUACCTUTAGAGUAU 6954-6974 A- 2814 UCAATACUCUAAAGGTAAG 6952-6974 1010690.1 1862528.1 UGA 1875228.1 UCUU AD- A- 2725 GGCGUUGUAGTUCCUAUC 2311-2331 A- 2815 UGAGAUAGGAACUACAAC 2309-2331 961202.1 1812640.1 UCA 1812641.1 GCCUU AD- A- 2726 UUUGUCGUAGTGAUUUU 1485-1505 A- 2816 UAGGAAAAUCACUACGACA 1483-1505 1010668.1 1852884.1 CCUA 1875206.1 AAGA AD- A- 2727 CAACUUACTUTCCUAAAU 7466-7486 A- 2817 UTAATUTAGGAAAGUAAGU 7464-7486 1010694.1 1863376.1 UAA 1875232.1 UGGU AD- A- 2728 GUAUGUUUCUAGCUGAU 5083-5103 A- 2818 UCAAAUCAGCUAGAAACAU 5081-5103 1010679.1 1859377.1 UUGA 1875217.1 ACCU AD- A- 2729 GACAGAGATGAUGAUUUA 6069-6089 A- 2819 UAGUAAAUCAUCATCTCUG 6067-6089 961257.1 1812750.1 CUA 1812751.1 UCUC AD- A- 2730 GAGAUGGATUCUCUUCG 5871-5891 A- 2820 UGAACGAAGAGAATCCAUC 5869-5891 961245.1 1812726.1 UUCA 1812727.1 UCCC AD- A- 2731 UUCCUGAUAUGCAGUUA 7259-7279 A- 2821 UAACTAACUGCAUAUCAGG 7257-7279 1010692.1 1863006.1 GUUA 1875230.1 AAGG AD- A- 2732 CACCUUCUCCTUAAAAUU 7537-7557 A- 2822 UAGAAUTUUAAGGAGAAG 7535-7557 1010695.1 1863481.1 CUA 1875233.1 GUGAC AD- A- 2733 CUGAUAAUAGTCUCUUAA 7161-7181 A- 2823 UGUUTAAGAGACUAUTAUC 7159-7181 961285.1 1812806.1 ACA 1812807.1 AGUA AD- A- 2734 CUAAAUUATGGAAGUAAU 7478-7498 A- 2824 UAGATUACUUCCATAAUUU 7476-7498 961300.1 1812836.1 CUA 1812837.1 AGGA AD- A- 2735 UCUUUAUACCAUCUUAG 8109-8129 A- 2825 UAACCUAAGAUGGTATAAA 8107-8129 961320.1 1812876.1 GUUA 1812877.1 GAAU AD- A- 2736 GGAGAUGGAUTCUCUUCG 5870-5890 A- 2826 UAACGAAGAGAAUCCAUCU 5868-5890 1010684.1 1860794.1 UUA 1875222.1 CCCC AD- A- 2737 UGAAUAUACAAGUAUUA 1676-1696 A- 2827 UTCCTAAUACUTGTATAUU 1674-1696 1010669.1 1853216.1 GGAA 1875207.1 CAGC AD- A- 2738 GUUUCUAGCUGAUUUGA 5087-5107 A- 2828 UCAATCAAAUCAGCUAGAA 5085-5107 1010680.1 1859383.1 UUGA 1875218.1 ACAU AD- A- 2739 AGUCAAGUTCCAAAUCGU 4392-4412 A- 2829 UGAACGAUUUGGAACTUG 4390-4412 961227.1 1812690.1 UCA 1812691.1 ACUUG AD- A- 2740 CAUCUGUUGGAAUAUUC 5506-5526 A- 2830 UGUAGAAUAUUCCAACAG 5504-5526 961243.1 1812722.1 UACA 1812723.1 AUGGG AD- A- 2741 CUGAACCUAUGAAUUCCG 3736-3756 A- 2831 UAUCGGAAUUCAUAGGUU 3734-3756 961221.1 1812678.1 AUA 1812679.1 CAGCC AD- A- 2742 CUUUUCACAGGAUUGUA 6586-6606 A- 2832 UAAUTACAAUCCUGUGAAA 6584-6606 961271.1 1812778.1 AUUA 1812779.1 AGAU AD- A- 2743 AGCGUGCUTATAGACGUU 5998-6018 A- 2833 UGUAACGUCUATAAGCACG 5996-6018 961251.1 1812738.1 ACA 1812739.1 CUGA AD- A- 2744 CUUCCUGATATGCAGUUA 7258-7278 A- 2834 UACUAACUGCATATCAGGA 7256-7278 961296.1 1812828.1 GUA 1812829.1 AGGA AD- A- 2745 GGAAGAAAGGTUCAUGUC 5897-5917 A- 2835 UCAGACAUGAACCTUTCUU 5895-5917 961246.1 1812728.1 UGA 1812729.1 CCAU AD- A- 2746 GUAGAAAACUTUUACAUC 6557-6577 A- 2836 UCAGAUGUAAAAGTUTUCU 6555-6577 1010688.1 1861826.1 UGA 1875226.1 ACAU AD- A- 2747 UCAUCUUUTCACAGGAUU 6582-6602 A- 2837 UACAAUCCUGUGAAAAGA 6580-6602 961269.1 1812774.1 GUA 1812775.1 UGACA AD- A- 2748 GCCCAAAATACUGAUAAU 7151-7171 A- 2838 UCUATUAUCAGTATUTUGG 7149-7171 1010691.1 1862804.1 AGA 1875229.1 GCAG AD- A- 2749 UUUUACAUCUGCCUUGU 6566-6586 A- 2839 UAUGACAAGGCAGAUGUA 6564-6586 1010689.1 1861844.1 CAUA 1875227.1 AAAGU AD- A- 2750 UAGGCUAATGACCCAAGA 1406-1426 A- 2840 UAAUCUTGGGUCATUAGCC 1404-1426 1010667.1 1852732.1 UUA 1875205.1 UAAA AD- A- 2751 CGUGCUUATAGACGUUAC 6000-6020 A- 2841 UCGGTAACGUCTATAAGCA 5998-6020 961252.1 1812740.1 CGA 1812741.1 CGCU AD- A- 2752 CUUCUUAGCCTUGUUUAG 1391-1411 A- 2842 UGCCTAAACAAGGCUAAGA 1389-1411 1010666.1 1852704.1 GCA 1875204.1 AGGC AD- A- 2753 UGGAAUAUTCTACUUUGU 5513-5533 A- 2843 UTAACAAAGUAGAAUAUUC 5511-5533 1010682.1 1860117.1 UAA 1875220.1 CAAC AD- A- 2754 CUGAAUAUACAAGUAUUA 1675-1695 A- 2844 UCCUAATACUUGUAUAUUC 1673-1695 961196.1 1812628.1 GGA 1812629.1 AGCC AD- A- 2755 CUGCCAAGTUAACAUAGA 3803-3823 A- 2845 UACUCUAUGUUAACUTGG 3801-3823 1010676.1 1857011.1 GUA 1875214.1 CAGCA AD- A- 2756 UGGAUUCUCUTCGUUCAC 5875-5895 A- 2846 UCUGTGAACGAAGAGAAUC 5873-5895 1010686.1 1860802.1 AGA 1875224.1 CAUC AD- A- 2757 GCAAAGGUCACAAUUUCC 3448-3468 A- 2847 UGAGGAAAUUGTGACCUU 3446-3468 1010675.1 1856353.1 UCA 1875213.1 UGCUC AD- A- 2758 UGCCACUGAAGAAAGUAC 5603-5623 A- 2848 UCAGTACUUUCTUCAGUGG 5601-5623 961244.1 1812724.1 UGA 1812725.1 CAAC AD- A- 2759 CCUUCCUGAUAUGCAGUU 7257-7277 A- 2849 UCUAACTGCAUAUCAGGAA 7255-7277 961295.1 1812826.1 AGA 1812827.1 GGAU AD- A- 2760 CAUCUUUUCACAGGAUU 6583-6603 A- 2850 UTACAATCCUGTGAAAAGA 6581-6603 961270.1 1812776.1 GUAA 1812777.1 UGAC AD- A- 2761 GGGAGAUGGATUCUCUUC 5869-5889 A- 2851 UACGAAGAGAATCCATCUC 5867-5889 1010683.1 1860792.1 GUA 1875221.1 CCCA AD- A- 2762 AAUGUCGGACTUGGUUAC 4479-4499 A- 2852 UAGGTAACCAAGUCCGACA 4477-4499 1010678.1 1858274.1 CUA 1875216.1 UUAU AD- A- 2763 UCAUCCUGGAAGUUCAGU 5468-5488 A- 2853 UCAACUGAACUTCCAGGAU 5466-5488 1010681.1 1860028.1 UGA 1875219.1 GAAC AD- A- 2764 AUGUAUAUTUGACCUAG 4826-4846 A- 2854 UTCACUAGGUCAAAUAUAC 4824-4846 961233.1 1812702.1 UGAA 1812703.1 AUCC AD- A- 2765 AGUCACCACUCAGCAUUC 1942-1962 A- 2855 UACGAATGCUGAGTGGUGA 1940-1962 961200.1 1812636.1 GUA 1812637.1 CUGA AD- A- 2766 AUCGUAAGAGAACUCUGU 6472-6492 A- 2856 UCUACAGAGUUCUCUTACG 6470-6492 961267.1 1812770.1 AGA 1812771.1 AUUC AD- A- 2767 GCUGAACCTATGAAUUCC 3735-3755 A- 2857 UTCGGAAUUCATAGGTUCA 3733-3755 961220.1 1812676.1 GAA 1812677.1 GCCU AD- A- 2768 CGGACUUGGUTACCUAUC 4484-4504 A- 2858 UGAGAUAGGUAACCAAGU 4482-4504 961232.1 1812700.1 UCA 1812701.1 CCGAC AD- A- 2769 AGAUGGAUTCTCUUCGUU 5872-5892 A- 2859 UTGAACGAAGAGAAUCCAU 5870-5892 1010685.1 1860796.1 CAA 1875223.1 CUCC AD- A- 2770 AGACGUUACCGCUUAAGG 6009-6029 A- 2860 UTGCCUTAAGCGGTAACGU 6007-6029 1010687.1 1861054.1 CAA 1875225.1 CUAU AD- A- 2771 UGUAGAUCTUGCAAUUAC 2543-2563 A- 2861 UTGGTAAUUGCAAGATCUA 2541-2563 961204.1 1812644.1 CAA 1812645.1 CAAA AD- A- 2772 UUGCCCUUAUGAAUGUU 4420-4440 A- 2862 UACUAACAUUCAUAAGGGC 4418-4440 961231.1 1812698.1 AGUA 1812699.1 AAAA

TABLE 6A Exemplary Human SCN9A siRNA Modified Single Strands and Duplex Sequences Column 1 indicates duplex name and the number following the decimal pointin a duplex name merely refers to a batch production number. Column 2 indicates the name of the sense sequence. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the modified sequence of a sense strand suitable for use in a duplex described herein. Column 5 indicates the antisense sequence name. Column 6 indicates the sequence ID for the sequence of column 7. Column 7 provides the sequence of a modified antisense strand suitable for use in a duplex described herein, e.g., a duplex comprising the sense sequence in the same row of the table. Column 8 indicates the position in the target mRNA (NM_001365536.1) that is complementary to the antisense strand of Column 7. Column 9 indicated the sequence ID for the sequence of column 8. Seq ID mRNA target SEQ ID Sense Seq ID Antisense NO: sequence in NO: Duplex sequence NO: Sense sequence sequence (anti Antisense sequence NM_0013 (mRNA Name name (sense) (5′-3′) name sense) (5′-3′) 65536.1 target) AD- A- 5816 gsgscgu(Uhd)GfuAf A- 5905 VPusGfsagau(Agn)gg AAGGCGUUGUAGU 3606 996318. 1525247.1 GfUfuccuaucucaL96 1240821.1 aacuAfcAfacgccsusu UCCUAUCUCC 1 AD- A- 5817 ususcug(Uhd)GfuAf A- 5906 VPusGfsugaa(Tgn)uc GCUUCUGUGUAGG 3607 995116. 1522818.1 GfGfagaauucacaL96 1238317.1 uccuAfcAfcagaasgsc AGAAUUCACU 1 AD- A- 5818 usgsguu(Uhd)CfaGf A- 5907 VPusCfsugaa(Tgn)cu UGUGGUUUCAGCA 3608 995486. 1523509.1 CfAfcagauucagaL96 1239063.1 gugcUfgAfaaccascsa CAGAUUCAGG 1 AD- A- 5819 usgsuag(Ghd)AfgAf A- 5908 VPusGfsaaaa(Ggn)ug UGUGUAGGAGAA 3609 995121. 1522828.1 AfUfucacuuuucaL96 1238327.1 aauuCfuCfcuacascsa UUCACUUUUCU 1 AD- A- 5820 ususugu(Ahd)GfaUf A- 5909 VPusGfsuaau(Tgn)gc CUUUUGUAGAUCU 3610 961022. 1525636.1 CfUfugcaauuacaL96 1241249.1 aagaUfcUfacaaasasg UGCAAUUACC 1 AD- A- 5821 gsusuug(Ahd)AfcAf A- 5910 VPusCfsgaaa(Ggn)au AGGUUUGAACACA 3611 1002051 1536779.1 CfAfaaucuuucgaL96 1252583.1 uuguGfuUfcaaacscs AAUCUUUCGG .1 u AD- A- 5822 csusucu(Ghd)AfaAf A- 5911 VPusCfsaguu(Tgn)gg UUCUUCUGAAACA 3612 995873. 1524297.1 CfAfuccaaacugaL96 1239861.1 auguUfuCfagaagsasa UCCAAACUGA 1 AD- A- 5823 asgsuca(Ahd)GfuUf A- 5912 VPusGfsaacg(Agn)uu CAAGUCAAGUUCC 3613 961040. 1529029.1 CfCfaaaucguucaL96 1244745.1 uggaAfcUfugacususg AAAUCGUUCC 1 AD- A- 5824 gsasucu(Uhd)CfuUf A- 5913 VPusUfscacu(Agn)cg AUGAUCUUCUUUG 3614 961013. 1523849.1 UfGfucguagugaaL96 1239411.1 acaaAfgAfagaucsasu UCGUAGUGAU 1 AD- A- 5825 usgsucg(Ahd)GfuAf A- 5914 VPusCfsagua(Agn)aa AAUGUCGAGUACA 3615 995055. 1522697.1 CfAfcuuuuacugaL96 1238195.1 guguAfcUfcgacasusu CUUUUACUGG 1 AD- A- 5826 csasuga(Uhd)CfuUf A- 5915 VPusCfsuacg(Agn)ca UACAUGAUCUUCU 3616 961010. 1523843.1 CfUfuugucguagaL96 1239405.1 aagaAfgAfucaugsusa UUGUCGUAGU 1 AD- A- 5827 asasggg(Ahd)AfaAf A- 5916 VPusAfscgga(Agn)ga CAAAGGGAAAACA 3617 961000. 1522351.1 CfAfaucuuccguaL96 1237849.1 uuguUfuUfcccuusus AUCUUCCGUU 1 g AD- A- 5828 asgsaug(Ghd)AfuUf A- 5917 VPusUfsgaac(Ggn)aa GGAGAUGGAUUCU 3618 999598. 1531657.1 CfUfcuucguucaaL96 1247453.1 gagaAfuCfcaucuscsc CUUCGUUCAC 1 AD- A- 5829 usgsaua(Ghd)UfuAf A- 5918 VPusUfsgcaa(Agn)cu UUUGAUAGUUACC 3619 1002101 1536879.1 CfCfuaguuugcaaL96 1252683.1 agguAfaCfuaucasasa UAGUUUGCAA .1 AD- A- 5830 usasuau(Uhd)UfuAf A- 5919 VPusAfsacgg(Agn)ug GAUAUAUUUUACA 3620 1001246 1535071.1 CfAfacauccguuaL96 1250879.1 uuguAfaAfauauasus ACAUCCGUUA .1 c AD- A- 5831 ususgcu(Ahd)UfaGf A- 5920 VPusGfsacca(Agn)au ACUUGCUAUAGGA 3621 996618. 1525802.1 GfAfaauuuggucaL96 1241423.1 uuccUfaUfagcaasgsu AAUUUGGUCU 1 AD- A- 5832 asuscuu(Chd)UfuUf A- 5921 VPusAfsucac(Tgn)ac UGAUCUUCUUUG 3622 961014. 1523851.1 GfUfcguagugauaL96 1239413.1 gacaAfaGfaagauscsa UCGUAGUGAUU 1 AD- A- 5833 asuscgu(Ahd)AfgAf A- 5922 VPusCfsuaca(Ggn)ag GAAUCGUAAGAGA 3623 1000046 1532577.1 GfAfacucuguagaL96 1248385.1 uucuCfuUfacgaususc ACUCUGUAGG .1 AD- A- 5834 gscsguu(Ghd)UfaGf A- 5923 VPusGfsgaga(Tgn)ag AGGCGUUGUAGU 3624 996319. 1525249.1 UfUfccuaucuccaL96 1240823.1 gaacUfaCfaacgcscsu UCCUAUCUCCU 1 AD- A- 5835 asusgau(Chd)UfuCf A- 5924 VPusAfscuac(Ggn)ac ACAUGAUCUUCUU 3625 961011. 1523845.1 UfUfugucguaguaL96 1239407.1 aaagAfaGfaucausgsu UGUCGUAGUG 1 AD- A- 5836 gscsugu(Uhd)UfaCf A- 5925 VPusAfsagaa(Tgn)cc AAGCUGUUUACAU 3626 1002409 1537499.1 AfUfaggauucuuaL96 1253305.1 uaugUfaAfacagcsusu AGGAUUCUUU .1 AD- A- 5837 csasccu(Uhd)CfuCfC A- 5926 VPusAfsgaau(Tgn)uu GUCACCUUCUCCU 3627 1000916 1534385.1 fUfuaaaauucuaL96 1250193.1 aaggAfgAfaggugsasc UAAAAUUCUA .1 AD- A- 5838 ususgug(Ahd)CfuUf A- 5927 VPusCfsacua(Agn)ac UAUUGUGACUUU 3628 996733. 1526036.1 UfAfaguuuagugaL96 1241657.1 uuaaAfgUfcacaasusa AAGUUUAGUGG 1 AD- A- 5839 uscsuuu(Ahd)UfaCf A- 5928 VPusAfsaccu(Agn)ag AUUCUUUAUACCA 3629 961137. 1535225.1 CfAfucuuagguuaL96 1251033.1 auggUfaUfaaagasas UCUUAGGUUC 1 u AD- A- 5840 gsasgau(Ghd)GfaUf A- 5929 VPusGfsaacg(Agn)ag GGGAGAUGGAUUC 3630 961057. 1531655.1 UfCfucuucguucaL96 1247451.1 agaaUfcCfaucucscsc UCUUCGUUCA 1 AD- A- 5841 ususgau(Ahd)GfuUf A- 5930 VPusGfscaaa(Cgn)ua UUUUGAUAGUUA 3631 1002100 1536877.1 AfCfcuaguuugcaL96 1252681.1 gguaAfcUfaucaasasa CCUAGUUUGCA .1 AD- A- 5842 gsascag(Ahd)GfaUf A- 5931 VPusAfsguaa(Agn)uc GAGACAGAGAUGA 3632 999762. 1531997.1 GfAfugauuuacuaL96 1247805.1 aucaUfcUfcugucsusc UGAUUUACUC 1 AD- A- 5843 asusgua(Chd)AfgAf A- 5932 VPusAfsuaga(Agn)ua CAAUGUACAGAGG 3633 961085. 1533099.1 GfGfuuauucuauaL9 1248907.1 accuCfuGfuacaususg UUAUUCUAUA 1 6 AD- A- 5844 asusguu(Uhd)CfuAf A- 5933 VPusAfsucaa(Agn)uc GUAUGUUUCUAGC 3634 961049. 1530270.1 GfCfugauuugauaL96 1246031.1 agcuAfgAfaacausasc UGAUUUGAUU 1 AD- A- 5845 csasaca(Chd)AfaUf A- 5934 VPusGfscuaa(Ggn)aa AACAACACAAUUU 3635 961155. 1535805.1 UfUfcuucuuagcaL96 1251613.1 gaaaUfuGfuguugsus CUUCUUAGCA 1 u AD- A- 5846 gscsaag(Uhd)CfaAf A- 5935 VPusCfsgauu(Tgn)gg CUGCAAGUCAAGU 3636 961039. 1529023.1 GfUfuccaaaucgaL96 1244739.1 aacuUfgAfcuugcsasg UCCAAAUCGU 1 AD- A- 5847 asasugu(Chd)GfgAf A- 5936 VPusAfsggua(Agn)cc AUAAUGUCGGACU 3637 998346. 1529197.1 CfUfugguuaccuaL96 1244919.1 aaguCfcGfacauusasu UGGUUACCUA 1 AD- A- 5848 csasucu(Ghd)UfuGf A- 5937 VPusGfsuaga(Agn)ua CCCAUCUGUUGGA 3638 961056. 1530988.1 GfAfauauucuacaL96 1246759.1 uuccAfaCfagaugsgsg AUAUUCUACU 1 AD- A- 5849 usgsgaa(Uhd)AfuUf A- 5938 VPusUfsaaca(Agn)ag GUUGGAAUAUUCU 3639 999259. 1531002.1 CfUfacuuuguuaaL96 1246773.1 uagaAfuAfuuccasasc ACUUUGUUAG 1 AD- A- 5850 csusgau(Ahd)AfuAf A- 5939 VPusGfsuuua(Agn)ga UACUGAUAAUAGU 3640 961093. 1533709.1 GfUfcucuuaaacaL96 1249517.1 gacuAfuUfaucagsusa CUCUUAAACU 1 AD- A- 5851 ususggc(Ahd)GfaAf A- 5940 VPusAfsuaau(Cgn)ag AAUUGGCAGAAAC 3641 995521. 1523579.1 AfCfccugauuauaL96 1239133.1 gguuUfcUfgccaasusu CCUGAUUAUG 1 AD- A- 5852 gscsaaa(Ghd)GfuCf A- 5941 VPusGfsagga(Agn)au GAGCAAAGGUCAC 3642 997386. 1527312.1 AfCfaauuuccucaL96 1242983.1 ugugAfcCfuuugcsusc AAUUUCCUCA 1 AD- A- 5853 csusgaa(Chd)CfuAf A- 5942 VPusAfsucgg(Agn)au GGCUGAACCUAUG 3643 961037. 1527831.1 UfGfaauuccgauaL96 1243513.1 ucauAfgGfuucagscsc AAUUCCGAUG 1 AD- A- 5854 gsgsaag(Ahd)AfaGf A- 5943 VPusCfsagac(Agn)ug AUGGAAGAAAGGU 3644 961058. 1531697.1 GfUfucaugucugaL96 1247503.1 aaccUfuUfcuuccsasu UCAUGUCUGC 1 AD- A- 5855 asgsccu(Ghd)UfuGf A- 5944 VPusAfsaacc(Tgn)au CAAGCCUGUUGGA 3645 961146. 1535441.1 GfAfaauagguuuaL96 1251249.1 uuccAfaCfaggcususg AAUAGGUUUU 1 AD- A- 5856 ususauu(Ghd)CfaUf A- 5945 VPusGfsuaua(Cgn)aa AUUUAUUGCAUCA 3646 1000747 1534041.1 CfAfcuuguauacaL96 1249849.1 gugaUfgCfaauaasasu CUUGUAUACA .1 AD- A- 5857 csusguu(Ghd)GfaAf A- 5946 VPusUfscaaa(Agn)cc GCCUGUUGGAAAU 3647 1001409 1535447.1 AfUfagguuuugaaL96 1251255.1 uauuUfcCfaacagsgsc AGGUUUUGAU .1 AD- A- 5858 asuscug(Ahd)GfaCf A- 5947 VPusCfsggca(Agn)au GGAUCUGAGACUG 3648 996130. 1524811.1 UfGfaauuugccgaL96 1240377.1 ucagUfcUfcagauscsc AAUUUGCCGA 1 AD- A- 5859 asgscgu(Ghd)CfuUf A- 5948 VPusGfsuaac(Ggn)uc UCAGCGUGCUUAU 3649 999715. 1531895.1 AfUfagacguuacaL96 1247701.1 uauaAfgCfacgcusgsa AGACGUUACC 1 AD- A- 5860 cscsuuc(Chd)UfgAf A- 5949 VPusCfsuaac(Tgn)gc AUCCUUCCUGAUA 3650 1000678 1533901.1 UfAfugcaguuagaL96 1249709.1 auauCfaGfgaaggsasu UGCAGUUAGU .1 AD- A- 5861 gsusaga(Ahd)AfaCf A- 5950 VPusCfsagau(Ggn)ua AUGUAGAAAACUU 3651 1000106 1532699.1 UfUfuuacaucugaL96 1248507.1 aaagUfuUfucuacsas UUACAUCUGC .1 u AD- A- 5862 gscscca(Ahd)AfaUfA A- 5951 VPusCfsuauu(Agn)uc CUGCCCAAAAUAC 3652 1000585 1533689.1 fCfugauaauagaL96 1249497.1 aguaUfuUfugggcsas UGAUAAUAGU .1 g AD- A- 5863 gsuscuu(Uhd)AfcUf A- 5952 VPusGfscaaa(Ggn)au UGGUCUUUACUG 3653 996635. 1525836.1 GfGfaaucuuugcaL96 1241457.1 uccaGfuAfaagacscsa GAAUCUUUGCA 1 AD- A- 5864 asgscuu(Ghd)AfaGf A- 5953 VPusGfsucua(Agn)uu UAAGCUUGAAGUA 3654 961163. 1536023.1 UfAfaaauuagacaL96 1251831.1 uuacUfuCfaagcususa AAAUUAGACC 1 AD- A- 5865 usgsgau(Uhd)CfuCf A- 5954 VPusCfsugug(Agn)ac GAUGGAUUCUCUU 3655 999601. 1531663.1 UfUfcguucacagaL96 1247459.1 gaagAfgAfauccasusc CGUUCACAGA 1 AD- A- 5866 ususuag(Uhd)GfgCf A- 5955 VPusCfsaaga(Ggn)ug ACUUUAGUGGCAA 3656 998015. 1528540.1 AfAfacacucuugaL96 1244249.1 uuugCfcAfcuaaasgsu ACACUCUUGG 1 AD- A- 5867 ascsaug(Ahd)UfcUf A- 5956 VPusUfsacga(Cgn)aa CUACAUGAUCUUC 3657 961009. 1523841.1 UfCfuuugucguaaL96 1239403.1 agaaGfaUfcaugusasg UUUGUCGUAG 1 AD- A- 5868 csasucu(Uhd)UfuCf A- 5957 VPusUfsacaa(Tgn)cc GUCAUCUUUUCAC 3658 961078. 1532751.1 AfCfaggauuguaaL96 1248559.1 ugugAfaAfagaugsasc AGGAUUGUAA 1 AD- A- 5869 csusgau(Uhd)UfcCf A- 5958 VPusCfsaccu(Tgn)uc CUCUGAUUUCCUA 3659 999986. 1532445.1 UfAfagaaaggugaL96 1248253.1 uuagGfaAfaucagsasg AGAAAGGUGG 1 AD- A- 5870 csusuua(Uhd)AfcCf A- 5959 VPusGfsaacc(Tgn)aa UUCUUUAUACCAU 3660 961138. 1535227.1 AfUfcuuagguucaL96 1251035.1 gaugGfuAfuaaagsas CUUAGGUUCA 1 a AD- A- 5871 csgsugc(Uhd)UfaUf A- 5960 VPusCfsggua(Agn)cg AGCGUGCUUAUAG 3661 961066. 1531899.1 AfGfacguuaccgaL96 1247705.1 ucuaUfaAfgcacgscsu ACGUUACCGC 1 AD- A- 5872 asasguc(Ahd)AfgUf A- 5961 VPusAfsacga(Tgn)uu GCAAGUCAAGUUC 3662 998261. 1529027.1 UfCfcaaaucguuaL96 1244743.1 ggaaCfuUfgacuusgsc CAAAUCGUUC 1 AD- A- 5873 csusgaa(Uhd)AfuAf A- 5962 VPusCfscuaa(Tgn)ac GGCUGAAUAUACA 3663 995823. 1524195.1 CfAfaguauuaggaL96 1239759.1 uuguAfuAfuucagscsc AGUAUUAGGA 1 AD- A- 5874 uscsgug(Ghd)CfuCf A- 5963 VPusCfsagaa(Agn)ac AUUCGUGGCUCCU 3664 996052. 1524655.1 CfUfuguuuucugaL96 1240221.1 aaggAfgCfcacgasasu UGUUUUCUGC 1 AD- A- 5875 asgsacg(Uhd)UfaCf A- 5964 VPusUfsgccu(Tgn)aa AUAGACGUUACCG 3665 999721. 1531917.1 CfGfcuuaaggcaaL96 1247723.1 gcggUfaAfcgucusasu CUUAAGGCAA 1 AD- A- 5876 uscsauc(Uhd)UfuUf A- 5965 VPusAfscaau(Cgn)cu UGUCAUCUUUUCA 3666 1000130 1532749.1 CfAfcaggauuguaL96 1248557.1 gugaAfaAfgaugascsa CAGGAUUGUA .1 AD- A- 5877 ususuua(Chd)AfuCf A- 5966 VPusAfsugac(Agn)ag ACUUUUACAUCUG 3667 1000115 1532717.1 UfGfccuugucauaL96 1248525.1 gcagAfuGfuaaaasgsu CCUUGUCAUC .1 AD- A- 5878 csusucc(Uhd)GfaUf A- 5967 VPusAfscuaa(Cgn)ug UCCUUCCUGAUAU 3668 961106. 1533903.1 AfUfgcaguuaguaL96 1249711.1 cauaUfcAfggaagsgsa GCAGUUAGUU 1 AD- A- 5879 usgsaau(Ahd)UfaCf A- 5968 VPusUfsccua(Agn)ua GCUGAAUAUACAA 3669 995824. 1524197.1 AfAfguauuaggaaL96 1239761.1 cuugUfaUfauucasgsc GUAUUAGGAG 1 AD- A- 5880 gsusuuc(Uhd)AfgCf A- 5969 VPusCfsaauc(Agn)aa AUGUUUCUAGCUG 3670 998897. 1530274.1 UfGfauuugauugaL9 1246035.1 ucagCfuAfgaaacsasu AUUUGAUUGA 1 6 AD- A- 5881 usgscca(Chd)UfgAf A- 5970 VPusCfsagua(Cgn)uu GUUGCCACUGAAG 3671 999348. 1531160.1 AfGfaaaguacugaL96 1246951.1 ucuuCfaGfuggcasasc AAAGUACUGA 1 AD- A- 5882 usgsauc(Uhd)UfcUf A- 5971 VPusCfsacua(Cgn)ga CAUGAUCUUCUUU 3672 961012. 1523847.1 UfUfgucguagugaL96 1239409.1 caaaGfaAfgaucasusg GUCGUAGUGA 1 AD- A- 5883 uscsauc(Chd)UfgGf A- 5972 VPusCfsaacu(Ggn)aa GUUCAUCCUGGAA 3673 999215. 1530912.1 AfAfguucaguugaL96 1246683.1 cuucCfaGfgaugasasc GUUCAGUUGA 1 AD- A- 5884 asusgua(Uhd)AfuUf A- 5973 VPusUfscacu(Agn)gg GGAUGUAUAUUU 3674 961044. 1529794.1 UfGfaccuagugaaL96 1245553.1 ucaaAfuAfuacauscsc GACCUAGUGAC 1 AD- A- 5885 asusguc(Ghd)AfgUf A- 5974 VPusAfsguaa(Agn)ag AAAUGUCGAGUAC 3675 961004. 1522695.1 AfCfacuuuuacuaL96 1238193.1 uguaCfuCfgacaususu ACUUUUACUG 1 AD- A- 5886 usasuug(Uhd)GfaCf A- 5975 VPusCfsuaaa(Cgn)uu CUUAUUGUGACUU 3676 961024. 1526032.1 UfUfuaaguuuagaL9 1241653.1 aaagUfcAfcaauasasg UAAGUUUAGU 1 6 AD- A- 5887 gsusaug(Uhd)UfuCf A- 5976 VPusCfsaaau(Cgn)ag AGGUAUGUUUCUA 3677 998894. 1530266.1 UfAfgcugauuugaL96 1246027.1 cuagAfaAfcauacscsu GCUGAUUUGA 1 AD- A- 5888 gsgsgag(Ahd)UfgGf A- 5977 VPusAfscgaa(Ggn)ag UGGGGAGAUGGA 3678 999596. 1531651.1 AfUfucucuucguaL96 1247447.1 aaucCfaUfcucccscsa UUCUCUUCGUU 1 AD- A- 5889 ususccu(Ghd)AfuAf A- 5978 VPusAfsacua(Agn)cu CCUUCCUGAUAUG 3679 1000679 1533905.1 UfGfcaguuaguuaL96 1249713.1 gcauAfuCfaggaasgsg CAGUUAGUUG .1 AD- A- 5890 csasacu(Uhd)AfcUf A- 5979 VPusUfsaauu(Tgn)ag ACCAACUUACUUU 3680 1000864 1534279.1 UfUfccuaaauuaaL96 1250087.1 gaaaGfuAfaguugsgs CCUAAAUUAU .1 u AD- A- 5891 usgscua(Uhd)AfgGf A- 5980 VPusAfsgacc(Agn)aa CUUGCUAUAGGAA 3681 996619. 1525804.1 AfAfauuuggucuaL96 1241425.1 uuucCfuAfuagcasasg AUUUGGUCUU 1 AD- A- 5892 csusaaa(Uhd)UfaUf A- 5981 VPusAfsgauu(Agn)cu UCCUAAAUUAUGG 3682 961109. 1534303.1 GfGfaaguaaucuaL96 1250111.1 uccaUfaAfuuuagsgsa AAGUAAUCUU 1 AD- A- 5893 gsascuu(Ahd)CfcUf A- 5982 VPusCfsaaua(Cgn)uc AAGACUUACCUUU 3683 1000451 1533415.1 UfUfagaguauugaL9 1249223.1 uaaaGfgUfaagucsus AGAGUAUUGU .1 6 u AD- A- 5894 csgsgac(Uhd)UfgGf A- 5983 VPusGfsagau(Agn)gg GUCGGACUUGGUU 3684 961043. 1529207.1 UfUfaccuaucucaL96 1244929.1 uaacCfaAfguccgsasc ACCUAUCUCU 1 AD- A- 5895 asgsuca(Chd)CfaCf A- 5984 VPusAfscgaa(Tgn)gc UCAGUCACCACUC 3685 996036. 1524627.1 UfCfagcauucguaL96 1240189.1 ugagUfgGfugacusgs AGCAUUCGUG 1 a AD- A- 5896 ususgcc(Chd)UfuAf A- 5985 VPusAfscuaa(Cgn)au UUUUGCCCUUAUG 3686 961042. 1529091.1 UfGfaauguuaguaL9 1244801.1 ucauAfaGfggcaasasa AAUGUUAGUC 1 6 AD- A- 5897 csusuuu(Chd)AfcAf A- 5986 VPusAfsauua(Cgn)aa AUCUUUUCACAGG 3687 1000133 1532757.1 GfGfauuguaauuaL9 1248565.1 uccuGfuGfaaaagsas AUUGUAAUUA .1 6 u AD- A- 5898 gscsuga(Ahd)CfcUf A- 5987 VPusUfscgga(Agn)uu AGGCUGAACCUAU 3688 961036. 1527829.1 AfUfgaauuccgaaL96 1243511.1 cauaGfgUfucagcscsu GAAUUCCGAU 1 AD- A- 5899 csusucu(Uhd)AfgCf A- 5988 VPusGfsccua(Agn)ac GCCUUCUUAGCCU 3689 995573. 1523683.1 CfUfuguuuaggcaL96 1239237.1 aaggCfuAfagaagsgsc UGUUUAGGCU 1 AD- A- 5900 csusgcc(Ahd)AfgUf A- 5989 VPusAfscucu(Agn)ug UGCUGCCAAGUUA 3690 997715. 1527964.1 UfAfacauagaguaL96 1243647.1 uuaaCfuUfggcagscsa ACAUAGAGUC 1 AD- A- 5901 usgsuag(Ahd)UfcUf A- 5990 VPusUfsggua(Agn)uu UUUGUAGAUCUU 3691 996533. 1525638.1 UfGfcaauuaccaaL96 1241253.1 gcaaGfaUfcuacasasa GCAAUUACCAU 1 AD- A- 5902 usasggc(Uhd)AfaUf A- 5991 VPusAfsaucu(Tgn)gg UUUAGGCUAAUGA 3692 995587. 1523713.1 GfAfcccaagauuaL96 1239267.1 gucaUfuAfgccuasasa CCCAAGAUUA 1 AD- A- 5903 ususugu(Chd)GfuAf A- 5992 VPusAfsggaa(Agn)au UCUUUGUCGUAG 3693 995660. 1523863.1 GfUfgauuuuccuaL96 1239425.1 cacuAfcGfacaaasgsa UGAUUUUCCUG 1 AD- A- 5904 ususgca(Ahd)GfcCf A- 5993 VPusCfsucac(Agn)ua GGUUGCAAGCCUC 3694 994670. 1521918.1 UfCfuuaugugagaL96 1237413.1 agagGfcUfugcaascsc UUAUGUGAGG 1

TABLE 6B Exemplary Human SCN9A Unmodified Single Strands and Duplex Sequences. Column 1 indicates duplex name and the number following the decimal point in a duplex name merely refers to a batch production number. Column 2 indicates the sense sequence name. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the unmodified sequence of a sense strand suitable for use in a duplex described herein. Column 5 provides the position in the target mRNA (NM_001365536.1) of the sense strand of Column 4. Column 6 indicates the antisense sequence name. Column 7 indicates the sequence ID for the sequence of column 8. Column 8 provides the sequence of an antisense strand suitable for use in a duplex described herein, without specifying chemical modifications. Column 9 indicates the position in the target mRNA (NM_001365536.1) that is complementary to the antisense strand of Column 8. Seq ID mRNA target Anti Seq ID mRNA target Sense NO: range in sense NO: range in Duplex sequence (sense) Sense NM_00136 sequence (anti antisense sequence NM_0013655 Name name sequence (5′-3′) 5536.1 name sense) (5′-3′) 36.1 AD- A- 5994 GGCGUUGUAGUUCCUAUC 2301-2321 A- 2950 UGAGAUAGGAACUACAAC 2299-2321 996318.1 1525247.1 UCA 1240821.1 GCCUU AD- A- 5995 UUCUGUGUAGGAGAAUU 824-844 A- 2951 UGUGAATUCUCCUACACA 822-844 995116.1 1522818.1 CACA 1238317.1 GAAGC AD- A- 2863 UGGUUUCAGCACAGAUUC 1243-1263 A- 2952 UCUGAATCUGUGCUGAAA 1241-1263 995486.1 1523509.1 AGA 1239063.1 CCACA AD- A- 2864 UGUAGGAGAAUUCACUU 829-849 A- 2953 UGAAAAGUGAAUUCUCCU 827-849 995121.1 1522828.1 UUCA 1238327.1 ACACA AD- A- 2865 UUUGUAGAUCUUGCAAU 2531-2551 A- 2954 UGUAAUTGCAAGAUCUAC 2529-2551 961022.1 1525636.1 UACA 1241249.1 AAAAG AD- A- 2866 GUUUGAACACAAAUCUUU 9174-9194 A- 2955 UCGAAAGAUUUGUGUUC 9172-9194 1002051.1 1536779.1 CGA 1252583.1 AAACCU AD- A- 2867 CUUCUGAAACAUCCAAAC 1683-1703 A- 2956 UCAGUUTGGAUGUUUCA 1681-1703 995873.1 1524297.1 UGA 1239861.1 GAAGAA AD- A- 2868 AGUCAAGUUCCAAAUCGU 4382-4402 A- 2957 UGAACGAUUUGGAACUU 4380-4402 961040.1 1529029.1 UCA 1244745.1 GACUUG AD- A- 2869 GAUCUUCUUUGUCGUAG 1435-1455 A- 2958 UUCACUACGACAAAGAAG 1433-1455 961013.1 1523849.1 UGAA 1239411.1 AUCAU AD- A- 2870 UGUCGAGUACACUUUUAC 760-780 A- 2959 UCAGUAAAAGUGUACUCG 758-780 995055.1 1522697.1 UGA 1238195.1 ACAUU AD- A- 2871 CAUGAUCUUCUUUGUCG 1432-1452 A- 2960 UCUACGACAAAGAAGAUC 1430-1452 961010.1 1523843.1 UAGA 1239405.1 AUGUA AD- A- 2872 AAGGGAAAACAAUCUUCC 576-596 A- 2961 UACGGAAGAUUGUUUUC 574-596 961000.1 1522351.1 GUA 1237849.1 CCUUUG AD- A- 2873 AGAUGGAUUCUCUUCGU 5862-5882 A- 2962 UUGAACGAAGAGAAUCCA 5860-5882 999598.1 1531657.1 UCAA 1247453.1 UCUCC AD- A- 2874 UGAUAGUUACCUAGUUU 9226-9246 A- 2963 UUGCAAACUAGGUAACUA 9224-9246 1002101.1 1536879.1 GCAA 1252683.1 UCAAA AD- A- 2875 UAUAUUUUACAACAUCCG 8022-8042 A- 2964 UAACGGAUGUUGUAAAA 8020-8042 1001246.1 1535071.1 UUA 1250879.1 UAUAUC AD- A- 2876 UUGCUAUAGGAAAUUUG 2625-2645 A- 2965 UGACCAAAUUUCCUAUAG 2623-2645 996618.1 1525802.1 GUCA 1241423.1 CAAGU AD- A- 2877 AUCUUCUUUGUCGUAGU 1436-1456 A- 2966 UAUCACTACGACAAAGAA 1434-1456 961014.1 1523851.1 GAUA 1239413.1 GAUCA AD- A- 2878 AUCGUAAGAGAACUCUGU 6462-6482 A- 2967 UCUACAGAGUUCUCUUAC 6460-6482 1000046.1 1532577.1 AGA 1248385.1 GAUUC AD- A- 2879 GCGUUGUAGUUCCUAUCU 2302-2322 A- 2968 UGGAGATAGGAACUACAA 2300-2322 996319.1 1525249.1 CCA 1240823.1 CGCCU AD- A- 2880 AUGAUCUUCUUUGUCGU 1433-1453 A- 2969 UACUACGACAAAGAAGAU 1431-1453 961011.1 1523845.1 AGUA 1239407.1 CAUGU AD- A- 2881 GCUGUUUACAUAGGAUUC 9600-9620 A- 2970 UAAGAATCCUAUGUAAAC 9598-9620 1002409.1 1537499.1 UUA 1253305.1 AGCUU AD- A- 2882 CACCUUCUCCUUAAAAUU 7527-7547 A- 2971 UAGAAUTUUAAGGAGAAG 7525-7547 1000916.1 1534385.1 CUA 1250193.1 GUGAC AD- A- 2883 UUGUGACUUUAAGUUUA 2742-2762 A- 2972 UCACUAAACUUAAAGUCA 2740-2762 996733.1 1526036.1 GUGA 1241657.1 CAAUA AD- A- 2884 UCUUUAUACCAUCUUAGG 8099-8119 A- 2973 UAACCUAAGAUGGUAUAA 8097-8119 961137.1 1535225.1 UUA 1251033.1 AGAAU AD- A- 2885 GAGAUGGAUUCUCUUCG 5861-5881 A- 2974 UGAACGAAGAGAAUCCAU 5859-5881 961057.1 1531655.1 UUCA 1247451.1 CUCCC AD- A- 2886 UUGAUAGUUACCUAGUU 9225-9245 A- 2975 UGCAAACUAGGUAACUAU 9223-9245 1002100.1 1536877.1 UGCA 1252681.1 CAAAA AD- A- 2887 GACAGAGAUGAUGAUUUA 6059-6079 A- 2976 UAGUAAAUCAUCAUCUCU 6057-6079 999762.1 1531997.1 CUA 1247805.1 GUCUC AD- A- 2888 AUGUACAGAGGUUAUUC 6778-6798 A- 2977 UAUAGAAUAACCUCUGUA 6776-6798 961085.1 1533099.1 UAUA 1248907.1 CAUUG AD- A- 2889 AUGUUUCUAGCUGAUUU 5075-5095 A- 2978 UAUCAAAUCAGCUAGAAA 5073-5095 961049.1 1530270.1 GAUA 1246031.1 CAUAC AD- A- 2890 CAACACAAUUUCUUCUUA 8498-8518 A- 2979 UGCUAAGAAGAAAUUGU 8496-8518 961155.1 1535805.1 GCA 1251613.1 GUUGUU AD- A- 2891 GCAAGUCAAGUUCCAAAU 4379-4399 A- 2980 UCGAUUTGGAACUUGACU 4377-4399 961039.1 1529023.1 CGA 1244739.1 UGCAG AD- A- 2892 AAUGUCGGACUUGGUUAC 4469-4489 A- 2981 UAGGUAACCAAGUCCGAC 4467-4489 998346.1 1529197.1 CUA 1244919.1 AUUAU AD- A- 2893 CAUCUGUUGGAAUAUUCU 5496-5516 A- 2982 UGUAGAAUAUUCCAACAG 5494-5516 961056.1 1530988.1 ACA 1246759.1 AUGGG AD- A- 2894 UGGAAUAUUCUACUUUG 5503-5523 A- 2983 UUAACAAAGUAGAAUAUU 5501-5523 999259.1 1531002.1 UUAA 1246773.1 CCAAC AD- A- 2895 CUGAUAAUAGUCUCUUAA 7151-7171 A- 2984 UGUUUAAGAGACUAUUA 7149-7171 961093.1 1533709.1 ACA 1249517.1 UCAGUA AD- A- 2896 UUGGCAGAAACCCUGAUU 1296-1316 A- 2985 UAUAAUCAGGGUUUCUG 1294-1316 995521.1 1523579.1 AUA 1239133.1 CCAAUU AD- A- 2897 GCAAAGGUCACAAUUUCC 3438-3458 A- 2986 UGAGGAAAUUGUGACCU 3436-3458 997386.1 1527312.1 UCA 1242983.1 UUGCUC AD- A- 2898 CUGAACCUAUGAAUUCCG 3726-3746 A- 2987 UAUCGGAAUUCAUAGGU 3724-3746 961037.1 1527831.1 AUA 1243513.1 UCAGCC AD- A- 2899 GGAAGAAAGGUUCAUGUC 5887-5907 A- 2988 UCAGACAUGAACCUUUCU 5885-5907 961058.1 1531697.1 UGA 1247503.1 UCCAU AD- A- 2900 AGCCUGUUGGAAAUAGG 8219-8239 A- 2989 UAAACCTAUUUCCAACAG 8217-8239 961146.1 1535441.1 UUUA 1251249.1 GCUUG AD- A- 2901 UUAUUGCAUCACUUGUA 7317-7337 A- 2990 UGUAUACAAGUGAUGCAA 7315-7337 1000747.1 1534041.1 UACA 1249849.1 UAAAU AD- A- 2902 CUGUUGGAAAUAGGUUU 8222-8242 A- 2991 UUCAAAACCUAUUUCCAA 8220-8242 1001409.1 1535447.1 UGAA 1251255.1 CAGGC AD- A- 2903 AUCUGAGACUGAAUUUGC 1993-2013 A- 2992 UCGGCAAAUUCAGUCUCA 1991-2013 996130.1 1524811.1 CGA 1240377.1 GAUCC AD- A- 2904 AGCGUGCUUAUAGACGUU 5988-6008 A- 2993 UGUAACGUCUAUAAGCAC 5986-6008 999715.1 1531895.1 ACA 1247701.1 GCUGA AD- A- 2905 CCUUCCUGAUAUGCAGUU 7247-7267 A- 2994 UCUAACTGCAUAUCAGGA 7245-7267 1000678.1 1533901.1 AGA 1249709.1 AGGAU AD- A- 2906 GUAGAAAACUUUUACAUC 6547-6567 A- 2995 UCAGAUGUAAAAGUUUU 6545-6567 1000106.1 1532699.1 UGA 1248507.1 CUACAU AD- A- 2907 GCCCAAAAUACUGAUAAU 7141-7161 A- 2996 UCUAUUAUCAGUAUUUU 7139-7161 1000585.1 1533689.1 AGA 1249497.1 GGGCAG AD- A- 2908 GUCUUUACUGGAAUCUU 2642-2662 A- 2997 UGCAAAGAUUCCAGUAAA 2640-2662 996635.1 1525836.1 UGCA 1241457.1 GACCA AD- A- 2909 AGCUUGAAGUAAAAUUAG 8687-8707 A- 2998 UGUCUAAUUUUACUUCA 8685-8707 961163.1 1536023.1 ACA 1251831.1 AGCUUA AD- A- 2910 UGGAUUCUCUUCGUUCAC 5865-5885 A- 2999 UCUGUGAACGAAGAGAAU 5863-5885 999601.1 1531663.1 AGA 1247459.1 CCAUC AD- A- 2911 UUUAGUGGCAAACACUCU 4114-4134 A- 3000 UCAAGAGUGUUUGCCACU 4112-4134 998015.1 1528540.1 UGA 1244249.1 AAAGU AD- A- 2912 ACAUGAUCUUCUUUGUCG 1431-1451 A- 3001 UUACGACAAAGAAGAUCA 1429-1451 961009.1 1523841.1 UAA 1239403.1 UGUAG AD- A- 2913 CAUCUUUUCACAGGAUUG 6573-6593 A- 3002 UUACAATCCUGUGAAAAG 6571-6593 961078.1 1532751.1 UAA 1248559.1 AUGAC AD- A- 2914 CUGAUUUCCUAAGAAAGG 6396-6416 A- 3003 UCACCUTUCUUAGGAAAU 6394-6416 999986.1 1532445.1 UGA 1248253.1 CAGAG AD- A- 2915 CUUUAUACCAUCUUAGGU 8100-8120 A- 3004 UGAACCTAAGAUGGUAUA 8098-8120 961138.1 1535227.1 UCA 1251035.1 AAGAA AD- A- 2916 CGUGCUUAUAGACGUUAC 5990-6010 A- 3005 UCGGUAACGUCUAUAAGC 5988-6010 961066.1 1531899.1 CGA 1247705.1 ACGCU AD- A- 2917 AAGUCAAGUUCCAAAUCG 4381-4401 A- 3006 UAACGATUUGGAACUUGA 4379-4401 998261.1 1529027.1 UUA 1244743.1 CUUGC AD- A- 2918 CUGAAUAUACAAGUAUUA 1632-1652 A- 3007 UCCUAATACUUGUAUAUU 1630-1652 995823.1 1524195.1 GGA 1239759.1 CAGCC AD- A- 2919 UCGUGGCUCCUUGUUUU 1915-1935 A- 3008 UCAGAAAACAAGGAGCCA 1913-1935 996052.1 1524655.1 CUGA 1240221.1 CGAAU AD- A- 2920 AGACGUUACCGCUUAAGG 5999-6019 A- 3009 UUGCCUTAAGCGGUAACG 5997-6019 999721.1 1531917.1 CAA 1247723.1 UCUAU AD- A- 2921 UCAUCUUUUCACAGGAUU 6572-6592 A- 3010 UACAAUCCUGUGAAAAGA 6570-6592 1000130.1 1532749.1 GUA 1248557.1 UGACA AD- A- 2922 UUUUACAUCUGCCUUGUC 6556-6576 A- 3011 UAUGACAAGGCAGAUGUA 6554-6576 1000115.1 1532717.1 AUA 1248525.1 AAAGU AD- A- 2923 CUUCCUGAUAUGCAGUUA 7248-7268 A- 3012 UACUAACUGCAUAUCAGG 7246-7268 961106.1 1533903.1 GUA 1249711.1 AAGGA AD- A- 2924 UGAAUAUACAAGUAUUAG 1633-1653 A- 3013 UUCCUAAUACUUGUAUA 1631-1653 995824.1 1524197.1 GAA 1239761.1 UUCAGC AD- A- 2925 GUUUCUAGCUGAUUUGA 5077-5097 A- 3014 UCAAUCAAAUCAGCUAGA 5075-5097 998897.1 1530274.1 UUGA 1246035.1 AACAU AD- A- 2926 UGCCACUGAAGAAAGUAC 5593-5613 A- 3015 UCAGUACUUUCUUCAGU 5591-5613 999348.1 1531160.1 UGA 1246951.1 GGCAAC AD- A- 2927 UGAUCUUCUUUGUCGUA 1434-1454 A- 3016 UCACUACGACAAAGAAGA 1432-1454 961012.1 1523847.1 GUGA 1239409.1 UCAUG AD- A- 2928 UCAUCCUGGAAGUUCAGU 5458-5478 A- 3017 UCAACUGAACUUCCAGGA 5456-5478 999215.1 1530912.1 UGA 1246683.1 UGAAC AD- A- 2929 AUGUAUAUUUGACCUAG 4816-4836 A- 3018 UUCACUAGGUCAAAUAUA 4814-4836 961044.1 1529794.1 UGAA 1245553.1 CAUCC AD- A- 2930 AUGUCGAGUACACUUUUA 759-779 A- 3019 UAGUAAAAGUGUACUCGA 757-779 961004.1 1522695.1 CUA 1238193.1 CAUUU AD- A- 2931 UAUUGUGACUUUAAGUU 2740-2760 A- 3020 UCUAAACUUAAAGUCACA 2738-2760 961024.1 1526032.1 UAGA 1241653.1 AUAAG AD- A- 2932 GUAUGUUUCUAGCUGAU 5073-5093 A- 3021 UCAAAUCAGCUAGAAACA 5071-5093 998894.1 1530266.1 UUGA 1246027.1 UACCU AD- A- 2933 GGGAGAUGGAUUCUCUU 5859-5879 A- 3022 UACGAAGAGAAUCCAUCU 5857-5879 999596.1 1531651.1 CGUA 1247447.1 CCCCA AD- A- 2934 UUCCUGAUAUGCAGUUAG 7249-7269 A- 3023 UAACUAACUGCAUAUCAG 7247-7269 1000679.1 1533905.1 UUA 1249713.1 GAAGG AD- A- 2935 CAACUUACUUUCCUAAAU 7456-7476 A- 3024 UUAAUUTAGGAAAGUAAG 7454-7476 1000864.1 1534279.1 UAA 1250087.1 UUGGU AD- A- 2936 UGCUAUAGGAAAUUUGG 2626-2646 A- 3025 UAGACCAAAUUUCCUAUA 2624-2646 996619.1 1525804.1 UCUA 1241425.1 GCAAG AD- A- 2937 CUAAAUUAUGGAAGUAAU 7468-7488 A- 3026 UAGAUUACUUCCAUAAUU 7466-7488 961109.1 1534303.1 CUA 1250111.1 UAGGA AD- A- 2938 GACUUACCUUUAGAGUAU 6944-6964 A- 3027 UCAAUACUCUAAAGGUAA 6942-6964 1000451.1 1533415.1 UGA 1249223.1 GUCUU AD- A- 2939 CGGACUUGGUUACCUAUC 4474-4494 A- 3028 UGAGAUAGGUAACCAAGU 4472-4494 961043.1 1529207.1 UCA 1244929.1 CCGAC AD- A- 2940 AGUCACCACUCAGCAUUC 1899-1919 A- 3029 UACGAATGCUGAGUGGUG 1897-1919 996036.1 1524627.1 GUA 1240189.1 ACUGA AD- A- 2941 UUGCCCUUAUGAAUGUUA 4410-4430 A- 3030 UACUAACAUUCAUAAGGG 4408-4430 961042.1 1529091.1 GUA 1244801.1 CAAAA AD- A- 2942 CUUUUCACAGGAUUGUAA 6576-6596 A- 3031 UAAUUACAAUCCUGUGAA 6574-6596 1000133.1 1532757.1 UUA 1248565.1 AAGAU AD- A- 2943 GCUGAACCUAUGAAUUCC 3725-3745 A- 3032 UUCGGAAUUCAUAGGUU 3723-3745 961036.1 1527829.1 GAA 1243511.1 CAGCCU AD- A- 2944 CUUCUUAGCCUUGUUUA 1348-1368 A- 3033 UGCCUAAACAAGGCUAAG 1346-1368 995573.1 1523683.1 GGCA 1239237.1 AAGGC AD- A- 2945 CUGCCAAGUUAACAUAGA 3793-3813 A- 3034 UACUCUAUGUUAACUUG 3791-3813 997715.1 1527964.1 GUA 1243647.1 GCAGCA AD- A- 2946 UGUAGAUCUUGCAAUUAC 2533-2553 A- 3035 UUGGUAAUUGCAAGAUC 2531-2553 996533.1 1525638.1 CAA 1241253.1 UACAAA AD- A- 2947 UAGGCUAAUGACCCAAGA 1363-1383 A- 3036 UAAUCUTGGGUCAUUAGC 1361-1383 995587.1 1523713.1 UUA 1239267.1 CUAAA AD- A- 2948 UUUGUCGUAGUGAUUUU 1442-1462 A- 3037 UAGGAAAAUCACUACGAC 1440-1462 995660.1 1523863.1 CCUA 1239425.1 AAAGA AD- A- 2949 UUGCAAGCCUCUUAUGUG 243-263 A- 3038 UCUCACAUAAGAGGCUUG 241-263 994670.1 1521918.1 AGA 1237413.1 CAACC

TABLE 13A Exemplary Human SCN9A siRNA Modified Single Strands and Duplex Sequences Column 1 indicates duplex name and the number following the decimal point in a duplex name merely refers to a batch production number. Column 2 indicates the name of the sense sequence. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the modified sequence of a sense strand suitable for use in a duplex described herein. Column 5 indicates the antisense sequence name. Column 6 indicates the sequence ID for the sequence of column 7. Column 7 provides the sequence of a modified antisense strand suitable for use in a duplex described herein, e.g., a duplex comprising the sense sequence in the same row of the table. Column 8 indicates the position in the target mRNA (NM_001365536.1) that is complementary to the antisense strand of Column 7. Column 9 indicated the sequence ID for the sequence of column 8. Seq ID mRNA target SEQ ID Sense Seq ID Antisense NO: Antisense sequence in NO: Duplex sequence NO: Sense sequence sequence (anti sequence NM_00136 (mRNA Name name (sense) (5′-3′) name sense) (5′-3′) 5536.1 target) AD- A- 4000 ascsacaaagdGgdAa A- 4266 VPusAfsgadTu(G2p) 1251302 2337487.1 aa(Chd)aaucuaL96 2337488.1 uuuudCcCfuUfugugu .1 susc AD- A- 5802 csascaaagggAfAfaac A- 4267 VPusAfsagdAudTguu 1251303 2337489.1 aa(Uhd)cuuaL96 2337490.1 uucCfcUfuugugsusu .1 AD- A- 5803 ascsaaagggAfAfAfac A- 4268 VPudGaadGadTuguu GAACAAAGGGAAA 4534 1251304 2337491.1 aa(Uhd)cuucaL96 2337492.1 uuCfcCfuuugusgsu ACAAUCUUCC .1 AD- A- 4003 csasaagggaAfAfAfca A- 4269 VPudGgadAgdAuugu AACAAAGGGAAAA 4535 1251305 2337493.1 au(Chd)uuccaL96 2337494.1 uuUfcCfcuuugsusg CAAUCUUCCG .1 AD- A- 4004 asasagggAfaAfAfCfa A- 4270 VPusCfsggdAadGauu ACAAAGGGAAAAC 4536 1251306 2337495.1 auc(Uhd)uccgaL96 2337496.1 guuUfuCfccuuusgsu AAUCUUCCGU .1 AD- A- 4005 asasagggaadAaCfaa A- 4271 VPuCfggdAadGauug ACAAAGGGAAAAC 4537 1251307 2337497.1 uc(Uhd)uccgaL96 2337498.1 dTuUfuCfccuuusgsu AAUCUUCCGU .1 AD- A- 4006 asasgggaaaAfCfAfa A- 4272 VPusdAscgdGa(A2p) CAAAGGGAAAACA 4538 1251315 2337506.1 ucu(Uhd)ccguaL96 2337501.1 gauudGuUfudTcccu AUCUUCCGUU .1 ususg AD- A- 4007 asasgggaaadAcdAa A- 4273 VPusdAscgdGa(A2p) CAAAGGGAAAACA 4539 1251310 2337499.1 ucu(Uhd)ccguaL96 2337501.1 gauudGuUfudTcccu AUCUUCCGUU .1 ususg AD- A- 4008 asasggg(Ahd)aadAc A- 4274 VPusdAscgdGadAga CAAAGGGAAAACA 4540 961179. 1812594.1 dAaucuuccguaL96 1812595.1 uudGudTudTcccuus AUCUUCCGUU 3 usg AD- A- 4009 asasgggaaadAcdAa A- 4275 VPusdAscgdGadAga CAAAGGGAAAACA 4541 1251308 2337499.1 ucu(Uhd)ccguaL96 1812595.1 uudGudTudTcccuus AUCUUCCGUU .1 usg AD- A- 4010 asasgggaaaAfCfAfa A- 4276 VPusdAscgdGa(Agn) CAAAGGGAAAACA 4542 1251314 2337506.1 ucu(Uhd)ccguaL96 2337500.1 gauudGuUfudTcccu AUCUUCCGUU .1 ususg AD- A- 4011 asasgggaaadAcdAa A- 4277 VPusdAscgdGa(Agn) CAAAGGGAAAACA 4543 1251309 2337499.1 ucu(Uhd)ccguaL96 2337500.1 gauudGuUfudTcccu AUCUUCCGUU .2 ususg AD- A- 4012 asasgggaaaAfCfAfa A- 4278 VPudAcgdGa(Agn)ga CAAAGGGAAAACA 4544 1251316 2337506.1 ucu(Uhd)ccguaL96 2337507.1 uudGuUfudTcccuus AUCUUCCGUU .1 usg AD- A- 4013 asasgggaaaAfCfAfa A- 4279 VPudAcgdGa(A2p)ga CAAAGGGAAAACA 4545 1251317 2337506.1 ucu(Uhd)ccguaL96 2337508.1 uudGuUfudTcccuus AUCUUCCGUU .1 usg AD- A- 4014 asasgggaaadAcdAa A- 4280 VPusdAscgdGa(A2p) CAAAGGGAAAACA 4546 1251311 2337499.1 ucu(Uhd)ccguaL96 2337502.1 gauudGuUfudTcccu AUCUUCCGUU .1 uscsc AD- A- 4015 asasgggaaadAcdAa A- 4281 VPusdAscgdGa(Agn) CAAAGGGAAAACA 4547 1251309 2337499.1 ucu(Uhd)ccguaL96 2337500.1 gauudGuUfudTcccu AUCUUCCGUU .1 ususg AD- A- 4016 asgsggaaAfaCfAfAfu A- 4282 VPusAfsacdGgdAaga AAAGGGAAAACAA 4548 1251318 2337509.1 cuu(Chd)cguuaL96 2337510.1 uugUfuUfucccususu UCUUCCGUUU .1 AD- A- 4017 asgsggaaaaCfadAuc A- 4283 VPudAacdGgdAagau AAAGGGAAAACAA 4549 1251319 2337511.1 uu(Chd)cguuaL96 2337512.1 dTgUfuUfucccususu UCUUCCGUUU .1 AD- A- 4018 gsgsgaaadAcdAauc A- 4284 VPusdAscgdGa(A2p) AAGGGAAAACAAU 4550 1251313 2337503.1 u(Uhd)ccguaL96 2337505.1 gauudGuUfudTcccsu CUUCCGUU .1 SU AD- A- 4019 gsgsgaaadAcdAauc A- 4285 VPusdAscgdGa(Agn) AAGGGAAAACAAU 4551 1251312 2337503.1 u(Uhd)ccguaL96 2337504.1 gauudGuUfudTcccsu CUUCCGUU .1 su AD- A- 4020 gsgsgaaaAfcAfaUfc A- 4286 VPusAfsaadCgdGaag AAGGGAAAACAAU 4552 1251320 2337513.1 uuc(Chd)guuuaL96 2337514.1 adTudGuUfuucccsus CUUCCGUUUC .1 u AD- A- 4021 gsgsaaaa(Chd)aaUf A- 4287 VPudGaadAcdGgaag AGGGAAAACAAUC 4553 1251321 2337515.1 CfuuccguuucaL96 2337516.1 auUfgUfuuuccscsu UUCCGUUUCA .1 AD- A- 4022 gsasaaa(Chd)aaUfCf A- 4288 VPuUfgadAa(C2p)gg GGGAAAACAAUCU 4554 1251323 2337519.1 UfuccguuucaaL96 2337520.1 aagaUfudGuuuucscs UCCGUUUCAA .1 c AD- A- 4023 gsasaaa(Chd)aaUfCf A- 4289 VPuUfgadAadTggaag GGGAAAACAAUCU 4555 1251322 2337517.1 UfuccauuucaaL96 2337518.1 aUfudGuuuucscsc UCCGUUUCAA .1 AD- A- 4024 asasaacaauCfUfUfc A- 4290 VPuUfugdAadAcgga GGAAAACAAUCUU 4556 1251325 2337523.1 cgu(Uhd)ucaaaL96 2337524.1 dAgdAuUfguuuuscsc CCGUUUCAAU .1 AD- A- 4025 asasaacaAfuCfUfUf A- 4291 VPusUfsugdAadAcgg GGAAAACAAUCUU 4557 1251324 2337521.1 ccgu(Uhd)ucaaaL96 2337522.1 aagAfuUfguuuuscsc CCGUUUCAAU .1 AD- A- 4026 usgsucgaguAfCfAfc A- 4292 VPusCfsagdTadAaag AAUGUCGAGUACA 4558 1251249 2337423.1 uuu(Uhd)acugaL96 2337424.1 uguAfcUfcgacasusu CUUUUACUGG .1 AD- A- 4027 usgsucgaguAfCfAfc A- 4293 VPuCfagdTadAaagug AAUGUCGAGUACA 4559 1251254 2337423.1 uuu(Uhd)acugaL96 2337431.1 uAfcUfcgacascsc CUUUUACUGG .1 AD- A- 4028 usgsucgaguAfCfAfc A- 4294 VPusCfsaguAfaAfAfg AAUGUCGAGUACA 4560 1251248 2337423.1 uuu(Uhd)acugaL96 1522698.1 uguAfcUfcgacasusu CUUUUACUGG .1 AD- A- 4029 usgsucgaguAfCfAfc A- 4295 VPusCfsagdTadAaag AAUGUCGAGUACA 4561 1251284 2337423.1 uuu(Uhd)acugaL96 2337467.1 udGuAfcdTcgacasus CUUUUACUGG .1 u AD- A- 4030 usgsucgagudAcdAc A- 4296 VPusCfsagdTadAaag AAUGUCGAGUACA 4562 1251253 2337428.1 uuu(Uhd)acugaL96 2337430.1 udGudAcUfcgacascs CUUUUACUGG .1 c AD- A- 4031 usgsucgaguAfCfAfc A- 4297 VPusCfsagdTadAaag AAUGUCGAGUACA 4563 1251286 2337423.1 uuu(Uhd)acugaL96 2337469.1 udGuAfcdTcgacascsc CUUUUACUGG .1 AD- A- 4032 usgsucgaguAfCfAfc A- 4298 VPusdCsagdTadAaag AAUGUCGAGUACA 4564 1251282 2337423.1 uuu(Uhd)acugaL96 1875199.1 udGudAcdTcgacasus CUUUUACUGG .1 u AD- A- 4033 usgsucg(Ahd)gudAc A- 4299 VPusdCsagdTadAaag AAUGUCGAGUACA 4565 1010661 1851664.1 dAcuuuuacugaL96 1875199.1 udGudAcdTcgacasus CUUUUACUGG .3 u AD- A- 4034 usgsucg(Ahd)GfuAf A- 4300 VPusCfsaguAfaAfAfg AAUGUCGAGUACA 4566 795305. 1522697.1 CfAfcuuuuacugaL96 1522698.1 uguAfcUfcgacasusu CUUUUACUGG 3 AD- A- 4035 usgsucgaguAfCfAfc A- 4301 VPusCfsagdTadAaag AAUGUCGAGUACA 4567 1251250 2337423.1 uuu(Uhd)acugaL96 2337425.1 uguAfcUfcgacascsc CUUUUACUGG .1 AD- A- 4036 usgsucgaguAfCfAfc A- 4302 VPusCfsagdTadAaag AAUGUCGAGUACA 4568 1251283 2337423.1 uuu(Uhd)acugaL96 2337466.1 udGudAcdTcgacasus CUUUUACUGG .1 u AD- A- 4037 usgsucgagudAcdAc A- 4303 VPusCfsagdTadAaag AAUGUCGAGUACA 4569 1251281 2337428.1 uuu(Uhd)acugaL96 2337466.1 udGudAcdTcgacasus CUUUUACUGG .1 u AD- A- 4038 usgsucgagudAcdAc A- 4304 VPuCfagdTadAaagud AAUGUCGAGUACA 4570 1251255 2337428.1 uuu(Uhd)acugaL96 2337432.1 GudAcUfcgacascsc CUUUUACUGG .1 AD- A- 4039 usgsucgagudAcdAc A- 4305 VPuCfagdTadAaagud AAUGUCGAGUACA 4571 1251289 2337428.1 uuu(Uhd)acugaL96 2337473.1 GudAcdTcgacasusu CUUUUACUGG .1 AD- A- 4040 usgsucgagudAcdAc A- 4306 VPusCfsagdTadAaag AAUGUCGAGUACA 4572 1251252 2337428.1 uuu(Uhd)acugaL96 2337429.1 udGudAcUfcgacasus CUUUUACUGG .1 u AD- A- 4041 usgsucgagudAcdAc A- 4307 VPusCfsagdTadAaag AAUGUCGAGUACA 4573 1251285 2337428.1 uuu(Uhd)acugaL96 2337468.1 udGudAcdTcgacascs CUUUUACUGG .1 c AD- A- 4042 usgsucgagudAcdAc A- 4308 VPuCfagdTadAaagud AAUGUCGAGUACA 4574 1251291 2337428.1 uuu(Uhd)acugaL96 2337475.1 GudAcdTcgacascsc CUUUUACUGG .1 AD- A- 4043 usgsucgaguAfCfAfc A- 4309 VPuCfagdTadAaagud AAUGUCGAGUACA 4575 1251290 2337423.1 uuu(Uhd)acugaL96 2337474.1 GuAfcdTcgacasusu CUUUUACUGG .1 AD- A- 4044 uscsgaguAfCfAfcuu A- 4310 VPusCfsagdTadAaag UGUCGAGUACACU 4576 1251251 2337426.1 u(Uhd)acugaL96 2337427.1 uguAfcUfcgascsg UUUACUGG .1 AD- A- 4045 uscsgagudAcdAcuu A- 4311 VPusCfsagdTadAaag UGUCGAGUACACU 4577 1251287 2337470.1 u(Uhd)acugaL96 2337471.1 udGudAcdTcgascsg UUUACUGG .1 AD- A- 4046 uscsgaguAfCfAfcuu A- 4312 VPusCfsagdTadAaag UGUCGAGUACACU 4578 1251288 2337426.1 u(Uhd)acugaL96 2337472.1 udGuAfcdTcgascsg UUUACUGG .1 AD- A- 4047 gsasggc(Uhd)UfcUf A- 4313 VPuUfucdTc(C2p)ua AAGAGGCUUCUGU 4579 1251326 2337525.1 gUfguaggagaaaL96 2337526.1 cadCadGaAfgccucsu GUAGGAGAAU .1 SU AD- A- 4048 asgsgcu(Uhd)cudGu A- 4314 VPudAuudCu(C2p)cu AGAGGCUUCUGUG 4580 1251327 1851778.1 dGuaggagaauaL96 2337527.1 acdAcdAgdAagccusc UAGGAGAAUU .1 SU AD- A- 4049 gsgscuu(Chd)UfgUf A- 4315 VPudAaudTc(Tgn)cc GAGGCUUCUGUGU 4581 1251328 2337528.1 gUfaggagaauuaL96 2337529.1 uadCaCfadGaagccsu AGGAGAAUUC .1 sc AD- A- 4050 gscsuuc(Uhd)gugUf A- 4316 VPudGaadTu(C2p)uc AGGCUUCUGUGUA 4582 1251329 2337530.1 AfggagaauucaL96 2337531.1 cuacAfcAfgaagcscsu GGAGAAUUCA .1 AD- A- 4051 csusucug(Uhd)gdTa A- 4317 VPuUfgadAu(Tgn)cu GGCUUCUGUGUAG 4583 1251330 2337532.1 dGgagaauucaaL96 2337533.1 ccdTaCfaCfagaagscs GAGAAUUCAC .1 c AD- A- 4052 ususcug(Uhd)GfuAf A- 4318 VPusGfsugaAfuUfCf GCUUCUGUGUAGG 4584 795366. 1522818.1 GfGfagaauucacaL96 1522819.1 uccuAfcAfcagaasgsc AGAAUUCACU 3 AD- A- 4053 ususcug(Uhd)GfuAf A- 4319 VPusGfsugdAa(Tgn) GCUUCUGUGUAGG 4585 1251331 1522818.1 GfGfagaauucacaL96 2337534.1 ucuccuAfcAfcagaasg AGAAUUCACU .1 sc AD- A- 4054 ususcug(Uhd)guAfg A- 4320 VPusdGsugdAa(U2p) GCUUCUGUGUAGG 4586 1251334 2337536.1 dGagaauucacaL96 2337538.1 ucucdCuAfcAfcagaas AGAAUUCACU .1 gsc AD- A- 4055 ususcug(Uhd)guAfg A- 4321 VPusdGsugdAa(Tgn) GCUUCUGUGUAGG 4587 1251333 2337536.1 dGagaauucacaL96 2337537.1 ucucdCuAfcAfcagaas AGAAUUCACU .1 gsc AD- A- 4056 ususcug(Uhd)gudAg A- 4322 VPudGugdAa(U2p)u GCUUCUGUGUAGG 4588 1251338 1851786.1 dGagaauucacaL96 2337542.1 cucdCudAcdAcagaas AGAAUUCACU .1 gsc AD- A- 4057 ususcug(Uhd)gudAg A- 4323 VPudGugdAa(Tgn)uc GCUUCUGUGUAGG 4589 1251337 1851786.1 dGagaauucacaL96 2337541.1 ucdCudAcdAcagaasg AGAAUUCACU .1 sc AD- A- 4058 ususcug(Uhd)guAfg A- 4324 VPusdGsugdAa(U2p) GCUUCUGUGUAGG 4590 1251336 2337536.1 dGagaauucacaL96 2337540.1 ucucdCuAfcAfcagaas AGAAUUCACU .1 use AD- A- 4059 ususcug(Uhd)guAfg A- 4325 VPusdGsugdAa(Tgn) GCUUCUGUGUAGG 4591 1251335 2337536.1 dGagaauucacaL96 2337539.1 ucucdCuAfcAfcagaas AGAAUUCACU .1 use AD- A- 4060 uscsuguguadGgdAg A- 4326 VPudAgudGa(Agn)u CUUCUGUGUAGGA 4592 1251339 2337543.1 aau(Uhd)cacuaL96 2337544.1 ucudCcUfaCfacagasg GAAUUCACUU .1 sg AD- A- 4061 csusgug(Uhd)agdGa A- 4327 VPudAagdTgdAauuc UUCUGUGUAGGA 4593 1251340 1851790.1 dGaauucacuuaL96 2337545.1 dTcCfudAcacagsgsg GAAUUCACUUU .1 AD- A- 4062 usgsug(Uhd)aggAfg A- 4328 VPudAaadGu(G2p)a UCUGUGUAGGAGA 4594 1251341 2337546.1 AfauucacuuuaL96 2337547.1 auudCuCfcUfacacas AUUCACUUUU .1 gsg AD- A- 4063 gsusguaggadGadAu A- 4329 VPudAaadAgdTgaau CUGUGUAGGAGAA 4595 1251342 2337548.1 uca(Chd)uuuuaL96 2337549.1 dTcUfcCfuacacsgsg UUCACUUUUC .1 AD- A- 4064 usgsuaggagdAaUfu A- 4330 VPusdGsaadAa(G2p) UGUGUAGGAGAA 4596 1251347 2337481.1 cac(Uhd)uuucaL96 2337555.1 ugaadTuCfuCfcuacas UUCACUUUUCU .1 esg AD- A- 4065 usgsuag(Ghd)AfgAf A- 4331 VPusGfsaaaAfgUfGfa UGUGUAGGAGAA 4597 795371. 1522828.1 AfUfucacuuuucaL96 1522829.1 auuCfuCfcuacascsa UUCACUUUUCU 3 AD- A- 4066 usgsuag(Ghd)agdAa A- 4332 VPusdGsaadAadGug UGUGUAGGAGAA 4598 1010663 1851796.1 dTucacuuuucaL96 1875201.1 aadTudCudCcuacasc UUCACUUUUCU .3 sa AD- A- 4067 usgsuaggagdAaUfU A- 4333 VPudGaadAa(G2p)u UGUGUAGGAGAA 4599 1251301 2337482.1 fcac(Uhd)uuucaL96 2337486.1 gaadTudCudCcuacas UUCACUUUUCU .1 csg AD- A- 4068 usgsuaggagdAaUfu A- 4334 VPusdGsaadAadAug UGUGUAGGAGAA 4600 1251348 2337556.1 cau(Uhd)uuucaL96 2337557.1 aadTuCfuCfcuacascs UUCACUUUUCU .1 g AD- A- 4069 usgsuaggAfgAfAfUf A- 4335 VPusGfsaaaAfgUfGfa UGUGUAGGAGAA 4601 1251343 2337550.1 ucac(Uhd)uuucaL96 1522829.1 auuCfuCfcuacascsa UUCACUUUUCU .1 AD- A- 4070 usgsuaggAfgAfAfUf A- 4336 VPusdGsaadAa(G2p) UGUAGGAGAAUUC 4602 1251346 2337550.1 ucac(Uhd)uuucaL96 2337554.1 ugaauuCfuCfcuascsg ACUUUUCU .1 AD- A- 4071 usgsuaggagdAadTu A- 4337 VPudGaadAa(G2p)u UGUGUAGGAGAA 4603 1251299 2337476.1 cac(Uhd)uuucaL96 2337486.1 gaadTudCudCcuacas UUCACUUUUCU .1 csg AD- A- 4072 usgsuaggAfgAfAfUf A- 4338 VPusdGsaadAadAug UGUGUAGGAGAA 4604 1251345 2337552.1 ucau(Uhd)uuucaL96 2337553.1 aauuCfuCfcuacascsg UUCACUUUUCU .1 AD- A- 4073 usgsuaggagdAaUfu A- 4339 VPudGaadAa(G2p)u UGUGUAGGAGAA 4605 1251349 2337481.1 cac(Uhd)uuucaL96 2337558.1 gaadTuCfuCfcuacasc UUCACUUUUCU .1 sg AD- A- 4074 usgsuaggagdAadTu A- 4340 VPusdGsaadAadGug UGUGUAGGAGAA 4606 1251292 2337476.1 cac(Uhd)uuucaL96 2337477.1 aadTudCudCcuacasc UUCACUUUUCU .1 sg AD- A- 4075 usgsuaggagdAadTu A- 4341 VPusdGsaadAa(G2p) UGUGUAGGAGAA 4607 1251293 2337476.1 cac(Uhd)uuucaL96 2337478.1 ugaadTudCudCcuac UUCACUUUUCU .1 ascsg AD- A- 4076 usgsuaggagdAadTu A- 4342 VPusdGsaadAadAug UGUGUAGGAGAA 4608 1251294 2337479.1 cau(Uhd)uuucaL96 2337480.1 aadTudCudCcuacasc UUCACUUUUCU .1 sg AD- A- 4077 usgsuaggAfgAfAfUf A- 4343 VPusdGsaadAa(G2p) UGUGUAGGAGAA 4609 1251344 2337550.1 ucac(Uhd)uuucaL96 2337551.1 ugaauuCfuCfcuacasc UUCACUUUUCU .1 sg AD- A- 4078 usgsuaggagdAaUfu A- 4344 VPudGaadAa(G2p)u UGUGUAGGAGAA 4610 1251300 2337481.1 cac(Uhd)uuucaL96 2337486.1 gaadTudCudCcuacas UUCACUUUUCU .1 csg AD- A- 4079 usgsuaggagdAaUfu A- 4345 VPusdGsaadAa(G2p) UGUGUAGGAGAA 4611 1251295 2337481.1 cac(Uhd)uuucaL96 2337478.1 ugaadTudCudCcuac UUCACUUUUCU .1 ascsg AD- A- 4080 usgsuaggagdAaUfu A- 4346 VPusdGsaadAa(G2p) UGUGUAGGAGAA 4612 1251296 2337482.1 fcac(Uhd)uuucaL96 2337478.1 ugaadTudCudCcuac UUCACUUUUCU .1 ascsg AD- A- 4081 gsusaggagaAfUfUfc A- 4347 VPusAfsgadAadAgug GUGUAGGAGAAU 4613 1251350 2337559.1 acu(Uhd)uucuaL96 2337560.1 aauUfcUfccuacsgsc UCACUUUUCUU .1 AD- A- 4082 gsusaggagaaUfUfca A- 4348 VPudAgadAadAguga GUGUAGGAGAAU 4614 1251351 2337561.1 cu(Uhd)uucuaL96 2337562.1 auUfcUfccuacsgsc UCACUUUUCUU .1 AD- A- 4083 usasggagaaUfUfCfa A- 4349 VPusdAsagdAadAag UGUAGGAGAAUUC 4615 1251353 2337565.1 cuu(Uhd)ucuuaL96 2337566.1 ugaaUfuCfuccuascsg ACUUUUCUUC .1 AD- A- 4084 usasggagAfaUfUfCf A- 4350 VPusAfsagdAadAagu UGUAGGAGAAUUC 4616 1251352 2337563.1 acuu(Uhd)ucuuaL96 2337564.1 gaaUfuCfuccuascsg ACUUUUCUUC .1 AD- A- 4085 usasggagdAaUfUfca A- 4351 VPusdGsaadAa(G2p) UGUAGGAGAAUUC 4617 1251298 2337485.1 c(Uhd)uuucaL96 2337484.1 ugaadTudCudCcuasc ACUUUUCU .1 sg AD- A- 4086 usasggagdAaUfucac A- 4352 VPusdGsaadAa(G2p) UGUAGGAGAAUUC 4618 1251297 2337483.1 (Uhd)uuucaL96 2337484.1 ugaadTudCudCcuasc ACUUUUCU .1 sg AD- A- 4087 asgsgagaauUfcdAcu A- 4353 VPudGaadGadAaagu GUAGGAGAAUUCA 4619 1251354 2337567.1 uu(Uhd)cuucaL96 2337568.1 dGaAfuUfcuccusgsc CUUUUCUUCG .1 AD- A- 4088 gsgsagaaUfuCfAfCf A- 4354 VPusCfsgadAgdAaaa UAGGAGAAUUCAC 4620 1251355 2337569.1 uuuu(Chd)uucgaL96 2337570.1 gugAfaUfucuccsusg UUUUCUUCGU .1 AD- A- 4089 gsgsagaaUfuCfaCfu A- 4355 VPuCfgadAgdAaaagd UAGGAGAAUUCAC 4621 1251356 2337571.1 uuu(Chd)uucgaL96 2337572.1 TgdAaUfucuccsusg UUUUCUUCGU .1 AD- A- 4090 gsasgaa(Uhd)UfcaC A- 4356 VPudAcgdAadGaaaa AGGAGAAUUCACU 4622 1251357 2337573.1 fUfuuucuucguaL96 2337574.1 dGudGadAuucucscs UUUCUUCGUG .1 u AD- A- 4091 cscsugaagcAfUfAfa A- 4357 VPusdGsaadAadCau AACCUGAAGCAUA 4623 1251358 2337575.1 aug(Uhd)uuucaL96 2337576.1 uudAudGcUfucaggs AAUGUUUUCG .1 USU AD- A- 4092 csusgaagCfaUfAfAf A- 4358 VPusCfsgadAadAcau ACCUGAAGCAUAA 4624 1251359 2337577.1 augu(Uhd)uucgaL96 2337578.1 uuaUfgCfuucagsgsu AUGUUUUCGA .1 AD- A- 4093 csusgaagcadTadAau A- 4359 VPuCfgadAadAcauu ACCUGAAGCAUAA 4625 1251360 2337579.1 gu(Uhd)uucgaL96 2337580.1 uaUfgCfuucagsgsu AUGUUUUCGA .1 AD- A- 4094 usgsaag(Chd)audAa A- 4360 VPuUfcgdAadAacau CCUGAAGCAUAAA 4626 1251361 1852317.1 dAuguuuucgaaL96 2337581.1 dTuAfudGcuucasgsg UGUUUUCGAA .1 AD- A- 4095 gsasagcauadAaUfgu A- 4361 VPuUfucdGadAaaca CUGAAGCAUAAAU 4627 1251363 2337584.1 uu(Uhd)cgaaaL96 2337585.1 dTuUfaUfgcuucsasg GUUUUCGAAA .1 AD- A- 4096 gsasagcaUfaAfAfUf A- 4362 VPusUfsucdGadAaac CUGAAGCAUAAAU 4628 1251362 2337582.1 guuu(Uhd)cgaaaL96 2337583.1 auuUfaUfgcuucsasg GUUUUCGAAA .1 AD- A- 4097 asasgca(Uhd)aadAu A- 4363 VPuUfuudCgdAaaac UGAAGCAUAAAUG 4629 1251364 1812604.1 dGuuuucgaaaaL96 2337586.1 dAuUfudAugcuuscsg UUUUCGAAAU .1 AD- A- 4098 asgscauaaaUfgUfuu A- 4364 VPudAuudTc(G2p)aa GAAGCAUAAAUGU 4630 1251372 2337591.1 u(Chd)gaaauaL96 2337598.1 aadCaUfuUfaugcusu UUUCGAAAUU .1 sc AD- A- 4099 asgscauaAfaUfGfUf A- 4365 VPusAfsuuuCfgAfAfa GAAGCAUAAAUGU 4631 1251366 2337589.1 uuu(Chd)gaaauaL96 1523300.1 acaUfuUfaugcususc UUUCGAAAUU .1 AD- A- 4100 asgscauaAfaUfGfUf A- 4366 VPusAfsuudTc(G2p)a GAAGCAUAAAUGU 4632 1251367 2337589.1 uuu(Chd)gaaauaL96 2337590.1 aaacaUfuUfaugcusu UUUCGAAAUU .1 sc AD- A- 4101 asgscau(Ahd)AfaUf A- 4367 VPusAfsuuuCfgAfAfa GAAGCAUAAAUGU 4633 795634. 1523299.1 GfUfuuucgaaauaL9 1523300.1 acaUfuUfaugcususc UUUCGAAAUU 4 6 AD- A- 4102 asgscauaaaUfgUfuu A- 4368 VPusAfsuudTcdAaaa GAAGCAUAAAUGU 4634 1251369 2337593.1 u(Uhd)gaaauaL96 2337594.1 adCaUfuUfaugcusus UUUCGAAAUU .1 c AD- A- 4103 asgscauaaaUfgUfuu A- 4369 VPusAfsuudTc(G2p)a GAAGCAUAAAUGU 4635 1251368 2337591.1 u(Chd)gaaauaL96 2337592.1 aaadCaUfuUfaugcus UUUCGAAAUU .1 use AD- A- 4104 asgscauaaaUfgUfuu A- 4370 VPudAuudTc(G2p)aa GAAGCAUAAAUGU 4636 1251373 2337591.1 u(Chd)gaaauaL96 2337599.1 aadCaUfuUfaugcusc UUUCGAAAUU .1 sc AD- A- 4105 asgsca(Uhd)aaaUfg A- 4371 VPudAuudTcdGaaaa GAAGCAUAAAUGU 4637 1251365 2337587.1 UfuuucgaaauaL96 2337588.1 dCaUfuUfaugcususc UUUCGAAAUU .1 AD- A- 4106 asgscauaaaUfgUfuu A- 4372 VPusdAsuudTc(G2p) GAAGCAUAAAUGU 4638 1251370 2337591.1 u(Chd)gaaauaL96 2337595.1 aaaadCaUfuUfaugcu UUUCGAAAUU .1 scsc AD- A- 4107 gscsa(Uhd)aaaugUf A- 4373 VPusdAsaudTu(C2p) AAGCAUAAAUGUU 4639 1251374 2337600.1 UfuucgaaauuaL96 2337601.1 gaaaacAfuUfuaugcs UUCGAAAUUC .1 usu AD- A- 4108 csasuaaa(Uhd)gUfU A- 4374 VPusdGsaadTu(Tgn) AGCAUAAAUGUUU 4640 1251375 2337602.1 fUfucgaaauucaL96 2337603.1 cgaaaaCfaUfuuaugsc UCGAAAUUCA .1 su AD- A- 4109 csasuaaaUfgUfuuu( A- 4375 VPusdAsuudTc(G2p) AGCAUAAAUGUUU 4641 1251371 2337596.1 Chd)gaaauaL96 2337597.1 aaaadCaUfuUfaugsc UCGAAAUU .1 su AD- A- 4110 asusaaa(Uhd)guUfU A- 4376 VPusUfsgadAudTucg GCAUAAAUGUUUU 4642 1251376 2337604.1 fUfcgaaauucaaL96 2337605.1 aaaAfcAfuuuausgsc CGAAAUUCAC .1 AD- A- 4111 asusaaa(Uhd)guUfU A- 4377 VPusUfsgadAudTucg GCAUAAAUGUUUU 4643 1251377 2337604.1 fUfcgaaauucaaL96 2337606.1 aaaAfcAfuuuausgsu CGAAAUUCAC .1 AD- A- 4112 usasaaugUfuUfuCfg A- 4378 VPudGugdAadTuucg CAUAAAUGUUUUC 4644 1251378 2337607.1 aaa(Uhd)ucacaL96 2337608.1 dAadAaCfauuuasusg GAAAUUCACU .1 AD- A- 4113 asasaug(Uhd)uuUfc A- 4379 VPudAgudGa(A2p)u AUAAAUGUUUUCG 4645 1251379 2337609.1 dGaaauucacuaL96 2337610.1 uucdGaAfaAfcauuus AAAUUCACUU .1 gsu AD- A- 4114 usasca(Uhd)gAfuCf A- 4380 VPusAfscgdAcdAaag CCUACAUGAUCUU 4646 1251380 2337611.1 UfUfcuuugucguaL96 2337612.1 aagAfuCfauguasgsg CUUUGUCGUA .1 AD- A- 4115 usasca(Uhd)gauCfU A- 4381 VPudAscgdAcdAaag CCUACAUGAUCUU 4647 1251381 2337613.1 fUfcuuugucguaL96 2337614.1 aagAfuCfauguascsc CUUUGUCGUA .1 AD- A- 4116 ascsaugaUfcUfUfCf A- 4382 VPuUfacdGa(C2p)aa CUACAUGAUCUUC 4648 1251382 2337615.1 uuug(Uhd)cguaaL96 2337616.1 agdAadGaUfcaugusg UUUGUCGUAG .1 sg AD- A- 4117 csasuga(Uhd)CfuUf A- 4383 VPuCfuadCgdAcaaad UACAUGAUCUUCU 4649 1251384 1523843.1 CfUfuugucguagaL96 2337457.1 GadAgdAucaugsusg UUGUCGUAGU .1 AD- A- 4118 csasuga(Uhd)cuUfC A- 4384 VPuCfuadCgdAcaaad UACAUGAUCUUCU 4650 1251274 2337449.1 fUfuugucguagaL96 2337457.1 GadAgdAucaugsusg UUGUCGUAGU .2 AD- A- 4119 csasuga(Uhd)cudTc A- 4385 VPusdCsuadCgdAcaa UACAUGAUCUUCU 4651 961188. 1812612.1 dTuugucguagaL96 1812613.1 adGadAgdAucaugsu UUGUCGUAGU 3 sa AD- A- 4120 csasuga(Uhd)CfuUf A- 4386 VPusCfsuadCgdAcaa UACAUGAUCUUCU 4652 1251383 1523843.1 CfUfuugucguagaL96 2337617.1 agaAfgAfucaugsusg UUGUCGUAGU .1 AD- A- 4121 csasuga(Uhd)cuUfC A- 4387 VPusCfsuadCgdAcaa UACAUGAUCUUCU 4653 1251269 2337449.1 fUfuugucguagaL96 2337451.1 adGadAgdAucaugsu UUGUCGUAGU .1 sg AD- A- 4122 csasuga(Uhd)cuUfC A- 4388 VPusdCsuadCgdAcaa UACAUGAUCUUCU 4654 1251270 2337449.1 fUfuugucguagaL96 2337452.1 adGadAgdAucaugscs UUGUCGUAGU .1 c AD- A- 4123 csasuga(Uhd)cuUfC A- 4389 VPusdCsuadCgdAcaa UACAUGAUCUUCU 4655 1251268 2337449.1 fUfuugucguagaL96 2337450.1 adGadAgdAucaugsu UUGUCGUAGU .1 sg AD- A- 4124 csasuga(Uhd)cuUfC A- 4390 VPuCfuadCgdAcaaad UACAUGAUCUUCU 4656 1251274 2337449.1 fUfuugucguagaL96 2337457.1 GadAgdAucaugsusg UUGUCGUAGU .1 AD- A- 4125 csasuga(Uhd)cuUfC A- 4391 VPusCfsuadCgdAcaa UACAUGAUCUUCU 4657 1251271 2337449.1 fUfuugucguagaL96 2337453.1 adGadAgdAucaugscs UUGUCGUAGU .1 c AD- A- 4126 csasuga(Uhd)cuUfC A- 4392 VPudCuadCgdAcaaa UACAUGAUCUUCU 4658 1251275 2337449.1 fUfuugucguagaL96 2337458.1 dGadAgdAucaugsus UUGUCGUAGU .2 g AD- A- 4127 csasuga(Uhd)cuUfC A- 4393 VPudCuadCgdAcaaa UACAUGAUCUUCU 4659 1251275 2337449.1 fUfuugucguagaL96 2337458.1 dGadAgdAucaugsus UUGUCGUAGU .1 g AD- A- 4128 asusgau(Chd)UfuCf A- 4394 VPudAcudAcdGacaa ACAUGAUCUUCUU 4660 1251385 1523845.1 UfUfugucguaguaL96 2337618.1 dAgdAadGaucausgs UGUCGUAGUG .1 u AD- A- 4129 usgsaucuUfCfUfuug A- 4395 VPusdCsuadCgdAcaa CAUGAUCUUCUUU 4661 1251272 2337454.1 u(Chd)guagaL96 2337455.1 adGadAgdAucasusg GUCGUAGU .1 AD- A- 4130 usgsauc(Uhd)UfcUf A- 4396 VPudCacdTadCgacad CAUGAUCUUCUUU 4662 1251386 1523847.1 UfUfgucguagugaL96 2337619.1 AadGadAgaucasusg GUCGUAGUGA .1 AD- A- 4131 usgsaucuUfCfUfuug A- 4397 VPusCfsuadCgdAcaa CAUGAUCUUCUUU 4663 1251273 2337454.1 u(Chd)guagaL96 2337456.1 adGadAgdAucasusg GUCGUAGU .1 AD- A- 4132 gsasucu(Uhd)CfuUf A- 4398 VPusUfscadCu(A2p)c AUGAUCUUCUUUG 4664 1251390 2337622.1 udGucguagugaaL96 2337624.1 gacdAaAfgAfagaucsg UCGUAGUGAU .1 su AD- A- 4133 gsasucu(Uhd)CfuUf A- 4399 VPuUfcadCu(A2p)cg AUGAUCUUCUUUG 4665 1251398 2337622.1 udGucguagugaaL96 2337630.1 acdAaAfgAfagaucsgs UCGUAGUGAU .1 u AD- A- 4134 gsasucu(Uhd)CfuUf A- 4400 VPusUfscadCu(A2p)c AUGAUCUUCUUUG 4666 1251396 2337629.1 UfgUfCfguagugaaL9 2337621.1 gacaaAfgAfagaucsgs UCGUAGUGAU .1 6 u AD- A- 4135 gsasucu(Uhd)CfuUf A- 4401 VPuUfcadCu(A2p)cg AUGAUCUUCUUUG 4667 1251399 2337628.1 udGUfcguagugaaL9 2337630.1 acdAaAfgAfagaucsgs UCGUAGUGAU .1 6 u AD- A- 4136 gsasucu(Uhd)CfuUf A- 4402 VPusUfscacUfaCfGfa AUGAUCUUCUUUG 4668 795913. 1523849.1 UfGfucguagugaaL96 1523850.1 caaAfgAfagaucsasu UCGUAGUGAU 3 AD- A- 4137 gsasucu(Uhd)CfuUf A- 4403 VPuUfcadCu(A2p)cg AUGAUCUUCUUUG 4669 1251400 2337629.1 UfgUfCfguagugaaL9 2337631.1 acaaAfgAfagaucsgsu UCGUAGUGAU .1 6 AD- A- 4138 gsasucu(Uhd)CfuUf A- 4404 VPusUfscadCu(A2p)c AUGAUCUUCUUUG 4670 1251388 1523849.1 UfGfucguagugaaL96 2337621.1 gacaaAfgAfagaucsgs UCGUAGUGAU .1 u AD- A- 4139 gsasucu(Uhd)cudTu A- 4405 VPusUfscadCu(A2p)c AUGAUCUUCUUUG 4671 1251397 1812618.1 dGucguagugaaL96 2337624.1 gacdAaAfgAfagaucsg UCGUAGUGAU .1 SU AD- A- 4140 gsasucu(Uhd)CfuUf A- 4406 VPusUfscadCu(A2p)c AUGAUCUUCUUUG 4672 1251395 2337628.1 udGUfcguagugaaL9 2337624.1 gacdAaAfgAfagaucsg UCGUAGUGAU .1 6 su AD- A- 4141 gsasucu(Uhd)CfuUf A- 4407 VPusUfscadCu(Agn)c AUGAUCUUCUUUG 4673 1251387 1523849.1 UfGfucguagugaaL96 2337620.1 gacaaAfgAfagaucsgs UCGUAGUGAU .1 u AD- A- 4142 gsasucu(Uhd)CfuUf A- 4408 VPusUfscadCu(Agn)c AUGAUCUUCUUUG 4674 1251389 2337622.1 udGucguagugaaL96 2337623.1 gacdAaAfgAfagaucsg UCGUAGUGAU .1 su AD- A- 4143 gsasucu(Uhd)CfuUf A- 4409 VPusUfscadCu(Agn)c AUGAUCUUCUUUG 4675 1251393 2337628.1 udGUfcguagugaaL9 2337623.1 gacdAaAfgAfagaucsg UCGUAGUGAU .1 6 su AD- A- 4144 gsasucu(Uhd)CfuUf A- 4410 VPusUfscadCu(Agn)c AUGAUCUUCUUUG 4676 1251394 2337629.1 UfgUfCfguagugaaL9 2337620.1 gacaaAfgAfagaucsgs UCGUAGUGAU .1 6 u AD- A- 4145 asuscuu(Chd)UfuUf A- 4411 VPudAsucdAc(Tgn)a UGAUCUUCUUUG 4677 1251401 2337632.1 gUfcguagugauaL96 2337633.1 cgadCaAfadGaagaus UCGUAGUGAUU .1 csg AD- A- 4146 uscsu(Uhd)CfuUfud A- 4412 VPusUfscadCu(Agn)c GAUCUUCUUUGUC 4678 1251391 2337625.1 GucguagugaaL96 2337626.1 gacdAaAfgAfagasusc GUAGUGAU .1 AD- A- 4147 uscsu(Uhd)CfuUfud A- 4413 VPusUfscadCu(A2p)c GAUCUUCUUUGUC 4679 1251392 2337625.1 GucguagugaaL96 2337627.1 gacdAaAfgAfagasusc GUAGUGAU .1 AD- A- 4148 uscsuuc(Uhd)UfugU A- 4414 VPudAaudCa(C2p)ua GAUCUUCUUUGUC 4680 1251402 2337634.1 fCfguagugauuaL96 2337635.1 cgacAfaAfgaagasusc GUAGUGAUUU .1 AD- A- 4149 csusucu(Uhd)ugUfc A- 4415 VPudAaadTc(A2p)cu AUCUUCUUUGUCG 4681 1251403 2337636.1 dGuagugauuuaL96 2337637.1 acdGaCfaAfagaagsgs UAGUGAUUUU .1 u AD- A- 4150 ususcuu(Uhd)guCfg A- 4416 VPudAaadAu(C2p)ac UCUUCUUUGUCGU 4682 1251404 2337638.1 UfagugauuuuaL96 2337639.1 uadCgAfcAfaagaasgs AGUGAUUUUC .1 g AD- A- 4151 uscsuuugUfcgUfAfg A- 4417 VPudGaadAadTcacu CUUCUUUGUCGUA 4683 1251405 2337640.1 uga(Uhd)uuucaL96 2337641.1 dAcdGaCfaaagasgsg GUGAUUUUCC .1 AD- A- 4152 asusccu(Uhd)UfugU A- 4418 VPusUfsgcdAa(G2p) GGAUCCUUUUGUA 4684 1251406 2337642.1 fAfgaucuugcaaL96 2337643.1 aucuacAfaAfaggausc GAUCUUGCAA .1 sc AD- A- 4153 uscscuu(Uhd)UfgUf A- 4419 VPusUfsugdCa(Agn) GAUCCUUUUGUAG 4685 1251407 2337644.1 adGaucuugcaaaL96 2337645.1 gaucdTaCfaAfaaggas AUCUUGCAAU .1 use AD- A- 4154 cscsuuu(Uhd)gudAg A- 4420 VPudAuudGc(A2p)ag AUCCUUUUGUAGA 4686 1251408 1854629.1 dAucuugcaauaL96 2337646.1 audCuAfcAfaaaggsgs UCUUGCAAUU .1 u AD- A- 4155 csusuuugUfagAfUfc A- 4421 VPusAfsaudTg(C2p)a UCCUUUUGUAGAU 4687 1251409 2337647.1 uug(Chd)aauuaL96 2337648.1 agaucUfaCfaaaagsgs CUUGCAAUUA .1 g AD- A- 4156 ususuug(Uhd)agAfU A- 4422 VPusUfsaadTu(G2p) 1251411 2337650.1 fCfuugcaauuaaL96 2337651.1 caagauCfuAfcaaagsc .1 sc AD- A- 4157 ususuug(Uhd)AfgAf A- 4423 VPusUfsaadTu(G2p) CCUUUUGUAGAUC 4689 1251410 1525635.1 UfCfuugcaauuaaL96 2337649.1 caagauCfuAfcaaaasg UUGCAAUUAC .1 sg AD- A- 4158 ususug(Uhd)agaUfC A- 4424 VPusGfsuaaUfuGfCf CUUUUGUAGAUCU 4690 1251412 2337652.1 fUfugcaauuacaL96 2337653.1 aagaUfcUfacaaasgsg UGCAAUUACC .1 AD- A- 4159 ususugu(Ahd)GfaUf A- 4425 VPusGfsuaaUfuGfCf CUUUUGUAGAUCU 4691 796825. 1525636.1 CfUfugcaauuacaL96 1257916.1 aagaUfcUfacaaasasg UGCAAUUACC 3 AD- A- 4160 ususug(Uhd)agaUfC A- 4426 VPusdGsuadAu(Tgn) CUUUUGUAGAUCU 4692 1251413 2337652.1 fUfugcaauuacaL96 2337654.1 gcaagaUfcUfacaaasg UGCAAUUACC .1 sg AD- A- 4161 ususug(Uhd)agaUfC A- 4427 VPusdGsuadAu(U2p) CUUUUGUAGAUCU 4693 1251414 2337652.1 fUfugcaauuacaL96 2337655.1 gcaagaUfcUfacaaasg UGCAAUUACC .1 sg AD- A- 4162 ususug(Uhd)agaUfC A- 4428 VPudGuadAu(Tgn)gc CUUUUGUAGAUCU 4694 1251415 2337652.1 fUfugcaauuacaL96 2337656.1 aagaUfcUfacaaasgsg UGCAAUUACC .1 AD- A- 4163 ususug(Uhd)agaUfC A- 4429 VPudGuadAu(U2p)g CUUUUGUAGAUCU 4695 1251416 2337652.1 fUfugcaauuacaL96 2337657.1 caagaUfcUfacaaasgs UGCAAUUACC .1 g AD- A- 4164 ususguagauCfUfUfg A- 4430 VPusdGsgudAa(U2p) UUUUGUAGAUCU 4696 1251417 2337658.1 caa(Uhd)uaccaL96 2337659.1 ugcaagAfuCfuacaasg UGCAAUUACCA .1 sg AD- A- 4165 usgsuagaUfcUfudG A- 4431 VPuUfggdTadAuugc UUUGUAGAUCUU 4697 1251418 2337660.1 caau(Uhd)accaaL96 2337661.1 dAadGaUfcuacasgsg GCAAUUACCAU .1 AD- A- 4166 gsusaga(Uhd)CfuUf A- 4432 VPudAugdGudAauug UUGUAGAUCUUGC 4698 1251419 2337662.1 gCfaauuaccauaL96 2337663.1 dCaAfgAfucuacsgsg AAUUACCAUU .1 AD- A- 4167 usasgau(Chd)UfugC A- 4433 VPusAfsaudGg(Tgn)a UGUAGAUCUUGCA 4699 1251420 2337664.1 fAfauuaccauuaL96 2337665.1 auugcAfadGaucuasc AUUACCAUUU .1 sg AD- A- 4168 asgsauc(Uhd)UfgCf A- 4434 VPusAfsaadTg(G2p)u GUAGAUCUUGCAA 4700 1251421 1525641.1 AfAfuuaccauuuaL96 2337666.1 aauugCfaAfgaucusgs UUACCAUUUG .1 C AD- A- 4169 asgsauc(Uhd)ugCfa A- 4435 VPudAaadTg(G2p)ua GUAGAUCUUGCAA 4701 1251422 2337667.1 dAuuaccauuuaL96 2337668.1 audTgCfadAgaucusg UUACCAUUUG .1 sc AD- A- 4170 usasaau(Uhd)audG A- 4436 VPudGgudTu(G2p)u AAUAAAUUAUGUG 4702 1251423 1856083.1 udGaaacaaaccaL96 2337669.1 uucdAcAfuAfauuuas AAACAAACCU .1 usu AD- A- 4171 asasuua(Uhd)gudG A- 4437 VPudAagdGu(U2p)u UAAAUUAUGUGAA 4703 1251425 1856087.1 adAacaaaccuuaL96 2337672.1 guudTcAfcAfuaauus ACAAACCUUA .1 usg AD- A- 4172 asusuaugugdAadAc A- 4438 VPuUfaadGg(Tgn)uu AAAUUAUGUGAAA 4704 1251427 2337675.1 aaa(Chd)cuuaaL96 2337676.1 gudTuCfaCfauaausu CAAACCUUAC .1 SU AD- A- 4173 asusuaugUfgAfAfAf A- 4439 VPusUfsaadGg(Tgn) AAAUUAUGUGAAA 4705 1251426 2337673.1 caaa(Chd)cuuaaL96 2337674.1 uuguuuCfaCfauaaus CAAACCUUAC .1 usu AD- A- 4174 ususaug(Uhd)gaAfA A- 4440 VPudGuadAg(G2p)u AAUUAUGUGAAAC 4706 1251428 2337677.1 fCfaaaccuuacaL96 2337678.1 uuguuUfcAfcauaasu AAACCUUACG .1 SU AD- A- 4175 usasugu(Ghd)AfaAf A- 4441 VPusCfsguaAfgGfUfu AUUAUGUGAAACA 4707 797564. 1527042.1 CfAfaaccuuacgaL96 1527043.1 uguUfuCfacauasasu AACCUUACGU 4 AD- A- 4176 usasugugAfaAfCfAf A- 4442 VPuCfgudAa(G2p)gu AUUAUGUGAAACA 4708 1251434 2337679.1 aacc(Uhd)uacgaL96 2337687.1 uuguUfuCfacauasgs AACCUUACGU .1 u AD- A- 4177 usasugugAfaAfCfAf A- 4443 VPusCfsgudAadAguu AUUAUGUGAAACA 4709 1251431 2337681.1 aacu(Uhd)uacgaL96 2337682.1 uguUfuCfacauasgsu AACCUUACGU .1 AD- A- 4178 usasugugaadAcdAa A- 4444 VPusCfsgudAa(G2p) AUUAUGUGAAACA 4710 1251433 2337685.1 acc(Uhd)uacgaL96 2337686.1 guuudGuUfuCfacaua AACCUUACGU .1 sgsu AD- A- 4179 usasugugAfaAfCfAf A- 4445 VPusCfsgudAa(G2p) AUUAUGUGAAACA 4711 1251430 2337679.1 aacc(Uhd)uacgaL96 2337680.1 guuuguUfuCfacauas AACCUUACGU .1 gsu AD- A- 4180 usasugugAfaAfCfAf A- 4446 VPusCfsguaAfgGfUfu AUUAUGUGAAACA 4712 1251429 2337679.1 aacc(Uhd)uacgaL96 1527043.1 uguUfuCfacauasasu AACCUUACGU .1 AD- A- 4181 usasugugaadAcdAa A- 4447 VPuCfgudAa(G2p)gu AUUAUGUGAAACA 4713 1251435 2337685.1 acc(Uhd)uacgaL96 2337688.1 uudGuUfuCfacauasg AACCUUACGU .1 SU AD- A- 4182 asusgugaAfaCfAfAf A- 4448 VPusAfscgdTa(A2p)g UUAUGUGAAACAA 4714 1251438 2337689.1 accu(Uhd)acguaL96 2337691.1 guuugUfuUfcacausg ACCUUACGUG .1 sg AD- A- 4183 asusgugaAfaCfAfAf A- 4449 VPusAfscguAfaGfGfu UUAUGUGAAACAA 4715 1251436 2337689.1 accu(Uhd)acguaL96 1527045.1 uugUfuUfcacausasa ACCUUACGUG .1 AD- A- 4184 asusgugaAfaCfAfAf A- 4450 VPusAfscgdTa(Agn)g UUAUGUGAAACAA 4716 1251437 2337689.1 accu(Uhd)acguaL96 2337690.1 guuugUfuUfcacausg ACCUUACGUG .1 sg AD- A- 4185 asusgug(Ahd)AfaCf A- 4451 VPusAfscguAfaGfGfu UUAUGUGAAACAA 4717 797565. 1527044.1 AfAfaccuuacguaL96 1527045.1 uugUfuUfcacausasa ACCUUACGUG 4 AD- A- 4186 asusgugaAfaCfAfAf A- 4452 VPuAfcgdTa(Agn)gg UUAUGUGAAACAA 4718 1251443 2337689.1 accu(Uhd)acguaL96 2337698.1 uuugUfuUfcacausgs ACCUUACGUG .1 g AD- A- 4187 asusgugaaaCfadAac A- 4453 VPudAcgdTa(A2p)gg UUAUGUGAAACAA 4719 1251444 2337695.1 cu(Uhd)acguaL96 2337699.1 uudTgUfuUfcacausg ACCUUACGUG .1 sg AD- A- 4188 asusgugaaaCfadAac A- 4454 VPusdAscgdTa(A2p) UUAUGUGAAACAA 4720 1251442 2337695.1 cu(Uhd)acguaL96 2337697.1 gguudTgUfuUfcacau ACCUUACGUG .1 sgsg AD- A- 4189 asusgugaaaCfadAac A- 4455 VPusdAscgdTa(Agn) UUAUGUGAAACAA 4721 1251441 2337695.1 cu(Uhd)acguaL96 2337696.1 gguudTgUfuUfcacau ACCUUACGUG .1 sgsg AD- A- 4190 usgsugaaacdAadAc A- 4456 VPusdCsacdGudAag UAUGUGAAACAAA 4722 1251445 2337700.1 cu(Uhd)acgugaL96 2337701.1 gudTudGuUfucacasu CCUUACGUGA .1 sg AD- A- 4191 gsusgaAfaCfAfAfacc A- 4457 VPusAfscgdTa(Agn)g AUGUGAAACAAAC 4723 1251439 2337692.1 u(Uhd)acguaL96 2337693.1 guuugUfuUfcacsgsu CUUACGUG .1 AD- A- 4192 gsusgaaaCfadAaCfc A- 4458 VPuUfcadCgdTaaggd AUGUGAAACAAAC 4724 1251447 2337704.1 uua(Chd)gugaaL96 2337705.1 TuUfgUfuucacsgsu CUUACGUGAA .1 AD- A- 4193 gsusgaaaCfaAfAfCfc A- 4459 VPusUfscadCgdTaag AUGUGAAACAAAC 4725 1251446 2337702.1 uua(Chd)gugaaL96 2337703.1 guuUfgUfuucacsgsu CUUACGUGAA .1 AD- A- 4194 usgsaaa(Chd)aaaCf A- 4460 VPusUfsucdAc(G2p) UGUGAAACAAACC 4726 1251448 2337706.1 CfuuacgugaaaL96 2337707.1 uaagguUfudGuuuca UUACGUGAAU .1 scsa AD- A- 4195 gsasaacaaaCfCfUfu A- 4461 VPusdAsuudCa(C2p) GUGAAACAAACCU 4727 1251450 2337710.1 acg(Uhd)gaauaL96 2337711.1 guaaggUfuUfguuucs UACGUGAAUU .1 asc AD- A- 4196 gsasaacaAfaCfCfUfu A- 4462 VPusAfsuudCa(C2p)g GUGAAACAAACCU 4728 1251449 2337708.1 acg(Uhd)gaauaL96 2337709.1 uaaggUfuUfguuucsa UACGUGAAUU .1 sc AD- A- 4197 asasacaaacCfUfUfac A- 4463 VPusdAsaudTc(A2p) 1251451 2337712.1 g(Uhd)gaauuaL96 2337713.1 cugadAgdGuUfuguu .1 uscsg AD- A- 4198 usgsuga(Uhd)auaUf A- 4464 VPudAugdTu(G2p)u AUUGUGAUAUAU 4730 1251453 2337716.1 UfuuacaacauaL96 2337717.1 aaaauAfuAfucacasgs UUUACAACAUC .1 u AD- A- 4199 usgsuga(Uhd)AfuAf A- 4465 VPusAfsugdTu(G2p) AUUGUGAUAUAU 4731 1251452 2337714.1 UfUfuuacaacauaL96 2337715.1 uaaaauAfuAfucacas UUUACAACAUC .1 gsu AD- A- 4200 gsusga(Uhd)aUfaUf A- 4466 VPudGaudGu(U2p)g UUGUGAUAUAUU 4732 1251454 2337718.1 UfUfuacaacaucaL96 2337719.1 uaaaaUfaUfaucacsgs UUACAACAUCC .1 g AD- A- 4201 usgsaua(Uhd)auUf A- 4467 VPudGgadTg(U2p)ug UGUGAUAUAUUU 4733 1251455 2337720.1 UfUfacaacauccaL96 2337721.1 uaaaAfuAfuaucascsg UACAACAUCCG .1 AD- A- 4202 gsasua(Uhd)aUfuUf A- 4468 VPusCfsggdAudGuug GUGAUAUAUUUU 4734 1251456 2337722.1 UfAfcaacauccgaL96 2337723.1 uaaAfaUfauaucsgsc ACAACAUCCGU .1 AD- A- 4203 gsasua(Uhd)aUfuUf A- 4469 VPuCfggdAudGuugu GUGAUAUAUUUU 4735 1251457 2337724.1 udAcaacauccgaL96 2337725.1 dAadAaUfauaucsgsc ACAACAUCCGU .1 AD- A- 4204 asusaua(Uhd)UfuUf A- 4470 VPudAcgdGadTguug UGAUAUAUUUUAC 4736 1251459 2337727.1 aCfaacauccguaL96 2337728.1 dTadAadAuauauscsg AACAUCCGUU .1 AD- A- 4205 asusaua(Uhd)UfuUf A- 4471 VPusAfscgdGadTguu UGAUAUAUUUUAC 4737 1251458 1535069.1 AfCfaacauccguaL96 2337726.1 guaAfaAfuauauscsg AACAUCCGUU .1 AD- A- 4206 usasuau(Uhd)UfuAf A- 4472 VPusAfsacdGg(Agn)u GAUAUAUUUUACA 4738 1251462 1535071.1 CfAfacauccguuaL96 2337731.1 guuguAfaAfauauasc ACAUCCGUUA .1 sc AD- A- 4207 usasuau(Uhd)UfuAf A- 4473 VPusAfsacdGg(A2p) GAUAUAUUUUACA 4739 1251461 1535071.1 CfAfacauccguuaL96 2337730.1 uguuguAfaAfauauas ACAUCCGUUA .1 use AD- A- 4208 usasuau(Uhd)UfuAf A- 4474 VPuAfacdGg(Agn)ug GAUAUAUUUUACA 4740 1251468 1535071.1 CfAfacauccguuaL96 2337738.1 uuguAfaAfauauascsc ACAUCCGUUA .1 AD- A- 4209 usasuau(Uhd)UfuAf A- 4475 VPusAfsacdGg(A2p) GAUAUAUUUUACA 4741 1251463 1535071.1 CfAfacauccguuaL96 2337732.1 uguuguAfaAfauauas ACAUCCGUUA .1 CSC AD- A- 4210 usasuau(Uhd)UfuAf A- 4476 VPusAfsacdGg(Agn)u GAUAUAUUUUACA 4742 1251460 1535071.1 CfAfacauccguuaL96 2337729.1 guuguAfaAfauauasu ACAUCCGUUA .1 sc AD- A- 4211 usasuau(Uhd)uudA A- 4477 VPudAacdGg(A2p)ug GAUAUAUUUUACA 4743 1251469 1864159.1 cdAacauccguuaL96 2337739.1 uudGudAadAauauas ACAUCCGUUA .1 use AD- A- 4212 usasuau(Uhd)UfuAf A- 4478 VPusAfsacgGfaUfGfu GAUAUAUUUUACA 4744 801647. 1535071.1 CfAfacauccguuaL96 1535072.1 uguAfaAfauauasusc ACAUCCGUUA 3 AD- A- 4213 usasuau(Uhd)uudA A- 4479 VPusdAsacdGg(A2p) GAUAUAUUUUACA 4745 1251467 1864159.1 cdAacauccguuaL96 2337737.1 uguudGudAadAauau ACAUCCGUUA .1 asusc AD- A- 4214 usasuau(Uhd)Ufud A- 4480 VPusdAsacdGg(A2p) GAUAUAUUUUACA 4746 1251466 2337736.1 AcdAacauccguuaL9 2337737.1 uguudGudAadAauau ACAUCCGUUA .1 6 asusc AD- A- 4215 asusauu(Uhd)UfaCf A- 4481 VPusUfsaadCgdGaug AUAUAUUUUACAA 4747 1251470 1535073.1 AfAfcauccguuaaL96 2337740.1 uugUfaAfaauausgsu CAUCCGUUAU .1 AD- A- 4216 asusauu(Uhd)UfaCf A- 4482 VPuUfaadCgdGaugu AUAUAUUUUACAA 4748 1251471 2337741.1 adAcauccguuaaL96 2337742.1 dTgUfadAaauausgsu CAUCCGUUAU .1 AD- A- 4217 usasu(Uhd)UfuAfCf A- 4483 VPusAfsacdGg(A2p) UAUAUUUUACAAC 4749 1251465 2337733.1 AfacauccguuaL96 2337735.1 uguuguAfaAfauasus AUCCGUUA .1 g AD- A- 4218 usasuuu(Uhd)acdA A- 4484 VPudAuadAcdGgaug UAUAUUUUACAAC 4750 1251472 2337743.1 aCfauccguuauaL96 2337744.1 dTudGudAaaauasus AUCCGUUAUU .1 g AD- A- 4219 usasu(Uhd)UfuAfCf A- 4485 VPusAfsacdGg(Agn)u UAUAUUUUACAAC 4751 1251464 2337733.1 AfacauccguuaL96 2337734.1 guuguAfaAfauasusg AUCCGUUA .1 AD- A- 4220 asusuuuaCfaAfCfAf A- 4486 VPusAfsaudAadCgga AUAUUUUACAACA 4752 1251473 2337745.1 uccg(Uhd)uauuaL96 2337746.1 uguUfgUfaaaausgsu UCCGUUAUUA .1 AD- A- 4221 asusuuuacadAcdAu A- 4487 VPudAaudAadCggau AUAUUUUACAACA 4753 1251474 2337747.1 ccg(Uhd)uauuaL96 2337748.1 dGuUfgUfaaaausgsu UCCGUUAUUA .1 AD- A- 4222 ususuua(Chd)aaCfa A- 4488 VPuUfaadTadAcggad UAUUUUACAACAU 4754 1251475 2337749.1 UfccguuauuaaL96 2337750.1 TgUfudGuaaaasusg CCGUUAUUAC .1 AD- A- 4223 ususua(Chd)aacaUf A- 4489 VPudGuadAudAacgg AUUUUACAACAUC 4755 1251476 2337751.1 CfcguuauuacaL96 2337752.1 dAudGuUfguaaasgs CGUUAUUACU .1 u AD- A- 4224 csasaca(Chd)aaUfUf A- 4490 VPudGcudAadGaaga AACAACACAAUUU 4756 1251279 2337459.1 UfcuucuuagcaL96 2337464.1 dAaUfudGuguugsus CUUCUUAGCA .1 u AD- A- 4225 csasaca(Chd)aaUfUf A- 4491 VPusdGscudAadGaa AACAACACAAUUU 4757 1251276 2337459.1 UfcuucuuagcaL96 2337460.1 gadAaUfudGuguugs CUUCUUAGCA .1 USU AD- A- 4226 csasaca(Chd)aaUfUf A- 4492 VPudGcudAadGaaga AACAACACAAUUU 4758 1251280 2337459.1 UfcuucuuagcaL96 2337465.1 dAaUfudGuguugscsc CUUCUUAGCA .1 AD- A- 4227 csasaca(Chd)aaUfUf A- 4493 VPusdGscudAadGaa AACAACACAAUUU 4759 1251277 2337459.1 UfcuucuuagcaL96 2337461.1 gadAaUfudGuguugs CUUCUUAGCA .1 CSC AD- A- 4228 csasaca(Chd)aadTu A- 4494 VPusdGscudAadGaa AACAACACAAUUU 4760 961334. 1812904.1 dTcuucuuagcaL96 1812905.1 gadAadTudGuguugs CUUCUUAGCA 3 USU AD- A- 4229 ascsacaaUfUfUfcuu A- 4495 VPusdGscudAadGaa CAACACAAUUUCU 4761 1251278 2337462.1 c(Uhd)uagcaL96 2337463.1 gadAaUfudGugusus UCUUAGCA .1 g AD- A- 4230 gsgscuu(Chd)aadGu A- 4496 VPuCfagdTadGgaacd CUGGCUUCAAGUG 4762 1251477 1865763.1 dGuuccuacugaL96 2337753.1 AcUfudGaagccsgsg UUCCUACUGU .1 AD- A- 4231 gscsuu(Chd)aagUfg A- 4497 VPudAcadGu(Agn)gg UGGCUUCAAGUGU 4763 1251478 2337754.1 UfuccuacuguaL96 2337755.1 aadCaCfuUfgaagcscs UCCUACUGUC .1 g AD- A- 4232 csusucaagugUfUfcc A- 4498 VPusdGsacdAg(Tgn) GGCUUCAAGUGUU 4764 1251479 2337756.1 ua(Chd)ugucaL96 2337757.1 aggaacAfcUfugaagsc CCUACUGUCA .1 sc AD- A- 4233 ususcaagUfgUfUfCf A- 4499 VPuUfgadCa(G2p)ua GCUUCAAGUGUUC 4765 1251481 2337758.1 cuac(Uhd)gucaaL96 2337760.1 ggaaCfaCfuugaasgsc CUACUGUCAU .1 AD- A- 4234 ususcaagUfgUfUfCf A- 4500 VPusUfsgadCa(G2p) GCUUCAAGUGUUC 4766 1251480 2337758.1 cuac(Uhd)gucaaL96 2337759.1 uaggaaCfaCfuugaasg CUACUGUCAU .1 sc AD- A- 4235 uscsaag(Uhd)guUfC A- 4501 VPusAfsugdAc(Agn)g CUUCAAGUGUUCC 4767 1251482 2337761.1 fCfuacugucauaL96 2337762.1 uaggaAfcAfcuugasgs UACUGUCAUG .1 g AD- A- 4236 uscsaag(Uhd)guUfC A- 4502 VPusdAsugdAc(A2p) CUUCAAGUGUUCC 4768 1251483 2337761.1 fCfuacugucauaL96 2337763.1 guagdGadAcdAcuug UACUGUCAUG .1 asgsg AD- A- 4237 csasagugUfuCfCfUf A- 4503 VPuCfaudGa(C2p)ag UUCAAGUGUUCCU 4769 1251492 2337764.1 acug(Uhd)caugaL96 2337773.1 uaggAfaCfacuugscsc ACUGUCAUGA .1 AD- A- 4238 csasagugUfuCfCfUf A- 4504 VPusCfsaudGadCagu UUCAAGUGUUCCU 4770 1251485 2337764.1 acug(Uhd)caugaL96 2337766.1 adGgdAaCfacuugsgs ACUGUCAUGA .1 g AD- A- 4239 csasagu(Ghd)UfuCf A- 4505 VPusCfsaugAfcAfGfu UUCAAGUGUUCCU 4771 802471. 1536717.1 CfUfacugucaugaL96 1536718.1 aggAfaCfacuugsasa ACUGUCAUGA 4 AD- A- 4240 csasagugUfuCfCfUf A- 4506 VPusCfsaugAfcAfGfu UUCAAGUGUUCCU 4772 1251486 2337764.1 acug(Uhd)caugaL96 1536718.1 aggAfaCfacuugsasa ACUGUCAUGA .1 AD- A- 4241 csasagugUfuCfCfUf A- 4507 VPusCfsaudGadCagu UUCAAGUGUUCCU 4773 1251484 2337764.1 acug(Uhd)caugaL96 2337765.1 aggAfaCfacuugsgsg ACUGUCAUGA .1 AD- A- 4242 csasagugUfuCfCfUf A- 4508 VPuCfaudGa(C2p)ag UUCAAGUGUUCCU 4774 1251491 2337764.1 acug(Uhd)caugaL96 2337772.1 uaggAfaCfacuugsgsg ACUGUCAUGA .1 AD- A- 4243 csasagugUfuCfCfUf A- 4509 VPusCfsaudGa(C2p) UUCAAGUGUUCCU 4775 1251487 2337764.1 acug(Uhd)caugaL96 2337767.1 aguaggAfaCfacuugsg ACUGUCAUGA .1 sg AD- A- 4244 csasagugUfuCfCfUf A- 4510 VPusCfsaudGa(C2p) UUCAAGUGUUCCU 4776 1251488 2337764.1 acug(Uhd)caugaL96 2337768.1 aguaggAfaCfacuugsc ACUGUCAUGA .1 sc AD- A- 4245 csasagugUfuCfCfUf A- 4511 VPusCfsaudGa(C2p) UUCAAGUGUUCCU 4777 1251490 2337764.1 acug(Uhd)caugaL96 2337771.1 aguadGgdAaCfacuug ACUGUCAUGA .1 sgsg AD- A- 4246 asasgug(Uhd)UfcCf A- 4512 VPuUfcadTg(A2p)ca UCAAGUGUUCCUA 4778 1251494 2337775.1 udAcugucaugaaL96 2337776.1 gudAgdGadAcacuus CUGUCAUGAC .1 gsg AD- A- 4247 asgsugUfuCfCfUfac A- 4513 VPuCfaudGa(C2p)ag CAAGUGUUCCUAC 4779 1251493 2337769.1 ug(Uhd)caugaL96 2337774.1 uaggAfaCfacususg UGUCAUGA .1 AD- A- 4248 asgsugUfuCfCfUfac A- 4514 VPusCfsaudGa(C2p) CAAGUGUUCCUAC 4780 1251489 2337769.1 ug(Uhd)caugaL96 2337770.1 aguaggAfaCfacususg UGUCAUGA .1 AD- A- 4249 asgsugu(Uhd)CfcUf A- 4515 VPudGucdAu(G2p)ac CAAGUGUUCCUAC 4781 1251495 2337777.1 aCfugucaugacaL96 2337778.1 agdTadGgdAacacusu UGUCAUGACC .1 sg AD- A- 4250 gsusguu(Chd)CfuaC A- 4516 VPudGgudCa(Tgn)ga AAGUGUUCCUACU 4782 1251496 2337779.1 fUfgucaugaccaL96 2337780.1 caguAfgdGaacacsus GUCAUGACCU .1 u AD- A- 4251 usgsuuc(Chd)UfaCf A- 4517 VPudAggdTc(Agn)ug AGUGUUCCUACUG 4783 1251497 2337781.1 udGUfcaugaccuaL9 2337782.1 acdAgUfadGgaacasc UCAUGACCUG .1 6 SU AD- A- 4252 gsusucc(Uhd)acUfg A- 4518 VPuCfagdGu(C2p)au GUGUUCCUACUGU 4784 1251498 2337783.1 UfcaugaccugaL96 2337784.1 gadCadGudAggaacs CAUGACCUGC .1 gsc AD- A- 4253 ususgau(Ahd)GfuUf A- 4519 VPusGfscaaAfcUfAfg UUUUGAUAGUUA 4785 802552. 1536877.1 AfCfcuaguuugcaL96 1536878.1 guaAfcUfaucaasasa CCUAGUUUGCA 3 AD- A- 4254 ususgauagudTadCc A- 4520 VPudGcadAadCuagg UUUUGAUAGUUA 4786 1251267 2337439.1 uag(Uhd)uugcaL96 2337448.1 dTadAcUfaucaasgsg CCUAGUUUGCA .1 AD- A- 4255 ususgauagudTadCc A- 4521 VPusdGscadAadCua UUUUGAUAGUUA 4787 1251260 2337439.1 uag(Uhd)uugcaL96 2337438.1 ggdTadAcUfaucaasg CCUAGUUUGCA .1 sg AD- A- 4256 ususgauaGfuUfAfCf A- 4522 VPusGfscaaAfcUfAfg UUUUGAUAGUUA 4788 1251256 2337433.1 cuag(Uhd)uugcaL96 1536878.1 guaAfcUfaucaasasa CCUAGUUUGCA .1 AD- A- 4257 ususgauaguUfAfCfc A- 4523 VPudGcadAadCuagg UUUUGAUAGUUA 4789 1251265 2337436.1 uag(Uhd)uugcaL96 2337447.1 uaAfcUfaucaasgsg CCUAGUUUGCA .1 AD- A- 4258 ususgau(Ahd)guUfA A- 4524 VPusdGscadAadCua UUUUGAUAGUUA 4790 1251257 2337434.1 fCfcuaguuugcaL96 2337435.1 gguaAfcUfaucaasgsg CCUAGUUUGCA .1 AD- A- 4259 ususgauaguUfaCfcu A- 4525 VPudGcadAadCuagg UUUUGAUAGUUA 4791 1251266 2337437.1 ag(Uhd)uugcaL96 2337448.1 dTadAcUfaucaasgsg CCUAGUUUGCA .1 AD- A- 4260 ususgauaguUfAfCfc A- 4526 VPusdGscadAadTua 1251264 2337445.1 uaa(Uhd)uugcaL96 2337446.1 gguaAfcUfaucaasgsg .1 AD- A- 4261 ususgauaguUfaCfcu A- 4527 VPusdGscadAadCua UUUUGAUAGUUA 4793 1251259 2337437.1 ag(Uhd)uugcaL96 2337438.1 ggdTadAcUfaucaasg CCUAGUUUGCA .1 sg AD- A- 4262 ususgauaguUfAfCfc A- 4528 VPusdGscadAadCua UUUUGAUAGUUA 4794 1251258 2337436.1 uag(Uhd)uugcaL96 2337435.1 gguaAfcUfaucaasgsg CCUAGUUUGCA .1 AD- A- 4263 gsasuagudTadCcua A- 4529 VPusdGscadAadCua UUGAUAGUUACCU 4795 1251263 2337444.1 g(Uhd)uugcaL96 2337443.1 ggdTadAcUfaucsgsg AGUUUGCA .1 AD- A- 4264 gsasuaguUfaCfcuag A- 4530 VPusdGscadAadCua UUGAUAGUUACCU 4796 1251262 2337442.1 (Uhd)uugcaL96 2337443.1 ggdTadAcUfaucsgsg AGUUUGCA .1 AD- A- 4265 gsasuaguUfAfCfcua A- 4531 VPusdGscadAadCua UUGAUAGUUACCU 4797 1251261 2337440.1 g(Uhd)uugcaL96 2337441.1 gguaAfcUfaucsgsg AGUUUGCA .1

TABLE 13B Exemplary Human SCN9A Unmodified Single Strands and Duplex Sequences. Column 1 indicates duplex name; the number following the decimal point in a duplex name merely refers to a batch production number. Column 2 indicates the sense sequence name. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the unmodified sequence of a sense strand suitable for use in a duplex described herein. Column 5 provides the position in the target mRNA (NM_001365536.1) of the sense strand of Column 4. Column 6 indicates the antisense sequence name. Column 7 indicates the sequence ID for the sequence of column 8. Column 8 provides the sequence of an antisense strand suitable for use in a duplex described herein, without specifying chemical modifications. Column 9 indicates the position in the target mRNA (NM_001365536.1) that is complementary to the antisense strand of Column 8. Sense Seq ID mRNA target Antisense mRNA target Duplex sequence NO: Sense sequence range in sequence Seq ID NO: antisense sequence range in Name name (sense) (5′-3′) NM_001365536.1 name (antisense) (5′-3′) NM_001365536.1 AD- A- 4798 ACACAAAGGGAAAAC  571-591 A- 5064 UAGATUGUUUUCCC 1251302.1 2337487.1 AAUCUA 2337488.1 UUUGUGUUC AD- A- 4799 CACAAAGGGAAAACA  572-592 A- 5065 UAAGAUTGUUUUCC 1251303.1 2337489.1 AUCUUA 2337490.1 CUUUGUGUU AD- A- 4800 ACAAAGGGAAAACAA  573-593 A- 5066 UGAAGATUGUUUU  571-593 1251304.1 2337491.1 UCUUCA 2337492.1 CCCUUUGUGU AD- A- 4801 CAAAGGGAAAACAAU  574-594 A- 5067 UGGAAGAUUGUUU  572-594 1251305.1 2337493.1 CUUCCA 2337494.1 UCCCUUUGUG AD- A- 4802 AAAGGGAAAACAAUC  575-595 A- 5068 UCGGAAGAUUGUU  573-595 1251306.1 2337495.1 UUCCGA 2337496.1 UUCCCUUUGU AD- A- 4803 AAAGGGAAAACAAUC  575-595 A- 5069 UCGGAAGAUUGTU  573-595 1251307.1 2337497.1 UUCCGA 2337498.1 UUCCCUUUGU AD- A- 4804 AAGGGAAAACAAUCU  576-596 A- 5070 UACGGAAGAUUGU  574-596 1251315.1 2337506.1 UCCGUA 2337501.1 UUTCCCUUUG AD- A- 4805 AAGGGAAAACAAUCU  576-596 A- 5071 UACGGAAGAUUGU  574-596 1251310.1 2337499.1 UCCGUA 2337501.1 UUTCCCUUUG AD- A- 4806 AAGGGAAAACAAUCU  576-596 A- 5072 UACGGAAGAUUGUT  574-596 961179.3 1812594.1 UCCGUA 1812595.1 UTCCCUUUG AD- A- 4807 AAGGGAAAACAAUCU  576-596 A- 5073 UACGGAAGAUUGUT  574-596 1251308.1 2337499.1 UCCGUA 1812595.1 UTCCCUUUG AD- A- 4808 AAGGGAAAACAAUCU  576-596 A- 5074 UACGGAAGAUUGU  574-596 1251314.1 2337506.1 UCCGUA 2337500.1 UUTCCCUUUG AD- A- 4809 AAGGGAAAACAAUCU  576-596 A- 5075 UACGGAAGAUUGU  574-596 1251309.2 2337499.1 UCCGUA 2337500.1 UUTCCCUUUG AD- A- 4810 AAGGGAAAACAAUCU  576-596 A- 5076 UACGGAAGAUUGU  574-596 1251316.1 2337506.1 UCCGUA 2337507.1 UUTCCCUUUG AD- A- 4811 AAGGGAAAACAAUCU  576-596 A- 5077 UACGGAAGAUUGU  574-596 1251317.1 2337506.1 UCCGUA 2337508.1 UUTCCCUUUG AD- A- 4812 AAGGGAAAACAAUCU  576-596 A- 5078 UACGGAAGAUUGU  574-596 1251311.1 2337499.1 UCCGUA 2337502.1 UUTCCCUUCC AD- A- 4813 AAGGGAAAACAAUCU  576-596 A- 5079 UACGGAAGAUUGU  574-596 1251309.1 2337499.1 UCCGUA 2337500.1 UUTCCCUUUG AD- A- 4814 AGGGAAAACAAUCU  577-597 A- 5080 UAACGGAAGAUUG  575-597 1251318.1 2337509.1 UCCGUUA 2337510.1 UUUUCCCUUU AD- A- 4815 AGGGAAAACAAUCU  577-597 A- 5081 UAACGGAAGAUTGU  575-597 1251319.1 2337511.1 UCCGUUA 2337512.1 UUUCCCUUU AD- A- 4816 GGGAAAACAAUCUU  578-596 A- 5082 UACGGAAGAUUGU  576-596 1251313.1 2337503.1 CCGUA 2337505.1 UUTCCCUU AD- A- 4817 GGGAAAACAAUCUU  578-596 A- 5083 UACGGAAGAUUGU  576-596 1251312.1 2337503.1 CCGUA 2337504.1 UUTCCCUU AD- A- 4818 GGGAAAACAAUCUU  578-598 A- 5084 UAAACGGAAGATUG  576-598 1251320.1 2337513.1 CCGUUUA 2337514.1 UUUUCCCUU AD- A- 4819 GGAAAACAAUCUUCC  579-599 A- 5085 UGAAACGGAAGAU  577-599 1251321.1 2337515.1 GUUUCA 2337516.1 UGUUUUCCCU AD- A- 4820 GAAAACAAUCUUCCG  580-600 A- 5086 UUGAAACGGAAGA  578-600 1251323.1 2337519.1 UUUCAA 2337520.1 UUGUUUUCCC AD- A- 4821 GAAAACAAUCUUCCA  580-600 A- 5087 UUGAAATGGAAGAU  578-600 1251322.1 2337517.1 UUUCAA 2337518.1 UGUUUUCCC AD- A- 4822 AAAACAAUCUUCCGU  581-601 A- 5088 UUUGAAACGGAAG  579-601 1251325.1 2337523.1 UUCAAA 2337524.1 AUUGUUUUCC AD- A- 4823 AAAACAAUCUUCCGU  581-601 A- 5089 UUUGAAACGGAAG  579-601 1251324.1 2337521.1 UUCAAA 2337522.1 AUUGUUUUCC AD- A- 4824 UGUCGAGUACACUU  760-780 A- 5090 UCAGTAAAAGUGUA  758-780 1251249.1 2337423.1 UUACUGA 2337424.1 CUCGACAUU AD- A- 4825 UGUCGAGUACACUU  760-780 A- 5091 UCAGTAAAAGUGUA  758-780 1251254.1 2337423.1 UUACUGA 2337431.1 CUCGACACC AD- A- 4826 UGUCGAGUACACUU  760-780 A- 5092 UCAGUAAAAGUGU  758-780 1251248.1 2337423.1 UUACUGA 1522698.1 ACUCGACAUU AD- A- 4827 UGUCGAGUACACUU  760-780 A- 5093 UCAGTAAAAGUGUA  758-780 1251284.1 2337423.1 UUACUGA 2337467.1 CTCGACAUU AD- A- 4828 UGUCGAGUACACUU  760-780 A- 5094 UCAGTAAAAGUGUA  758-780 1251253.1 2337428.1 UUACUGA 2337430.1 CUCGACACC AD- A- 4829 UGUCGAGUACACUU  760-780 A- 5095 UCAGTAAAAGUGUA  758-780 1251286.1 2337423.1 UUACUGA 2337469.1 CTCGACACC AD- A- 4830 UGUCGAGUACACUU  760-780 A- 5096 UCAGTAAAAGUGUA  758-780 1251282.1 2337423.1 UUACUGA 1875199.1 CTCGACAUU AD- A- 4831 UGUCGAGUACACUU  803-823 A- 5097 UCAGTAAAAGUGUA  801-823 1010661.3 1851664.1 UUACUGA 1875199.1 CTCGACAUU AD- A- 4832 UGUCGAGUACACUU  760-780 A- 5098 UCAGUAAAAGUGU  758-780 795305.3 1522697.1 UUACUGA 1522698.1 ACUCGACAUU AD- A- 4833 UGUCGAGUACACUU  760-780 A- 5099 UCAGTAAAAGUGUA  758-780 1251250.1 2337423.1 UUACUGA 2337425.1 CUCGACACC AD- A- 4834 UGUCGAGUACACUU  760-780 A- 5100 UCAGTAAAAGUGUA  758-780 1251283.1 2337423.1 UUACUGA 2337466.1 CTCGACAUU AD- A- 4835 UGUCGAGUACACUU  760-780 A- 5101 UCAGTAAAAGUGUA  758-780 1251281.1 2337428.1 UUACUGA 2337466.1 CTCGACAUU AD- A- 4836 UGUCGAGUACACUU  760-780 A- 5102 UCAGTAAAAGUGUA  758-780 1251255.1 2337428.1 UUACUGA 2337432.1 CUCGACACC AD- A- 4837 UGUCGAGUACACUU  760-780 A- 5103 UCAGTAAAAGUGUA  758-780 1251289.1 2337428.1 UUACUGA 2337473.1 CTCGACAUU AD- A- 4838 UGUCGAGUACACUU  760-780 A- 5104 UCAGTAAAAGUGUA  758-780 1251252.1 2337428.1 UUACUGA 2337429.1 CUCGACAUU AD- A- 4839 UGUCGAGUACACUU  760-780 A- 5105 UCAGTAAAAGUGUA  758-780 1251285.1 2337428.1 UUACUGA 2337468.1 CTCGACACC AD- A- 4840 UGUCGAGUACACUU  760-780 A- 5106 UCAGTAAAAGUGUA  758-780 1251291.1 2337428.1 UUACUGA 2337475.1 CTCGACACC AD- A- 4841 UGUCGAGUACACUU  760-780 A- 5107 UCAGTAAAAGUGUA  758-780 1251290.1 2337423.1 UUACUGA 2337474.1 CTCGACAUU AD- A- 4842 UCGAGUACACUUUU  762-780 A- 5108 UCAGTAAAAGUGUA  760-780 1251251.1 2337426.1 ACUGA 2337427.1 CUCGACG AD- A- 4843 UCGAGUACACUUUU  762-780 A- 5109 UCAGTAAAAGUGUA  760-780 1251287.1 2337470.1 ACUGA 2337471.1 CTCGACG AD- A- 4844 UCGAGUACACUUUU  762-780 A- 5110 UCAGTAAAAGUGUA  760-780 1251288.1 2337426.1 ACUGA 2337472.1 CTCGACG AD- A- 4845 GAGGCUUCUGUGUA  819-839 A- 5111 UUUCTCCUACACAG  817-839 1251326.1 2337525.1 GGAGAAA 2337526.1 AAGCCUCUU AD- A- 4846 AGGCUUCUGUGUAG  863-883 A- 5112 UAUUCUCCUACACA  861-883 1251327.1 1851778.1 GAGAAUA 2337527.1 GAAGCCUCU AD- A- 4847 GGCUUCUGUGUAGG  821-841 A- 5113 UAAUTCTCCUACAC  819-841 1251328.1 2337528.1 AGAAUUA 2337529.1 AGAAGCCUC AD- A- 4848 GCUUCUGUGUAGGA  822-842 A- 5114 UGAATUCUCCUACA  820-842 1251329.1 2337530.1 GAAUUCA 2337531.1 CAGAAGCCU AD- A- 4849 CUUCUGUGTAGGAG  823-843 A- 5115 UUGAAUTCUCCTAC  821-843 1251330.1 2337532.1 AAUUCAA 2337533.1 ACAGAAGCC AD- A- 4850 UUCUGUGUAGGAGA  824-844 A- 5116 UGUGAAUUCUCCU  822-844 795366.3 1522818.1 AUUCACA 1522819.1 ACACAGAAGC AD- A- 4851 UUCUGUGUAGGAGA  824-844 A- 5117 UGUGAATUCUCCUA  822-844 1251331.1 1522818.1 AUUCACA 2337534.1 CACAGAAGC AD- A- 4852 UUCUGUGUAGGAGA  824-844 A- 5118 UGUGAAUUCUCCU  822-844 1251334.1 2337536.1 AUUCACA 2337538.1 ACACAGAAGC AD- A- 4853 UUCUGUGUAGGAGA  824-844 A- 5119 UGUGAATUCUCCUA  822-844 1251333.1 2337536.1 AUUCACA 2337537.1 CACAGAAGC AD- A- 4854 UUCUGUGUAGGAGA  867-887 A- 5120 UGUGAAUUCUCCU  865-887 1251338.1 1851786.1 AUUCACA 2337542.1 ACACAGAAGC AD- A- 4855 UUCUGUGUAGGAGA  867-887 A- 5121 UGUGAATUCUCCUA  865-887 1251337.1 1851786.1 AUUCACA 2337541.1 CACAGAAGC AD- A- 4856 UUCUGUGUAGGAGA  824-844 A- 5122 UGUGAAUUCUCCU  822-844 1251336.1 2337536.1 AUUCACA 2337540.1 ACACAGAAUC AD- A- 4857 UUCUGUGUAGGAGA  824-844 A- 5123 UGUGAATUCUCCUA  822-844 1251335.1 2337536.1 AUUCACA 2337539.1 CACAGAAUC AD- A- 4858 UCUGUGUAGGAGAA  825-845 A- 5124 UAGUGAAUUCUCC  823-845 1251339.1 2337543.1 UUCACUA 2337544.1 UACACAGAGG AD- A- 4859 CUGUGUAGGAGAAU  869-889 A- 5125 UAAGTGAAUUCTCC  867-889 1251340.1 1851790.1 UCACUUA 2337545.1 UACACAGGG AD- A- 4860 UGUGUAGGAGAAUU  827-847 A- 5126 UAAAGUGAAUUCU  825-847 1251341.1 2337546.1 CACUUUA 2337547.1 CCUACACAGG AD- A- 4861 GUGUAGGAGAAUUC  828-848 A- 5127 UAAAAGTGAAUTCU  826-848 1251342.1 2337548.1 ACUUUUA 2337549.1 CCUACACGG AD- A- 4862 UGUAGGAGAAUUCA  829-849 A- 5128 UGAAAAGUGAATUC  827-849 1251347.1 2337481.1 CUUUUCA 2337555.1 UCCUACACG AD- A- 4863 UGUAGGAGAAUUCA  829-849 A- 5129 UGAAAAGUGAAUU  827-849 795371.3 1522828.1 CUUUUCA 1522829.1 CUCCUACACA AD- A- 4864 UGUAGGAGAATUCA  872-892 A- 5130 UGAAAAGUGAATUC  870-892 1010663.3 1851796.1 CUUUUCA 1875201.1 UCCUACACA AD- A- 4865 UGUAGGAGAAUUCA  829-849 A- 5131 UGAAAAGUGAATUC  827-849 1251301.1 2337482.1 CUUUUCA 2337486.1 UCCUACACG AD- A- 4866 UGUAGGAGAAUUCA  829-849 A- 5132 UGAAAAAUGAATUC  827-849 1251348.1 2337556.1 UUUUUCA 2337557.1 UCCUACACG AD- A- 4867 UGUAGGAGAAUUCA  829-849 A- 5133 UGAAAAGUGAAUU  827-849 1251343.1 2337550.1 CUUUUCA 1522829.1 CUCCUACACA AD- A- 4868 UGUAGGAGAAUUCA  829-849 A- 5134 UGAAAAGUGAAUU  829-849 1251346.1 2337550.1 CUUUUCA 2337554.1 CUCCUACG AD- A- 4869 UGUAGGAGAATUCA  829-849 A- 5135 UGAAAAGUGAATUC  827-849 1251299.1 2337476.1 CUUUUCA 2337486.1 UCCUACACG AD- A- 4870 UGUAGGAGAAUUCA  829-849 A- 5136 UGAAAAAUGAAUUC  827-849 1251345.1 2337552.1 UUUUUCA 2337553.1 UCCUACACG AD- A- 4871 UGUAGGAGAAUUCA  829-849 A- 5137 UGAAAAGUGAATUC  827-849 1251349.1 2337481.1 CUUUUCA 2337558.1 UCCUACACG AD- A- 4872 UGUAGGAGAATUCA  829-849 A- 5138 UGAAAAGUGAATUC  827-849 1251292.1 2337476.1 CUUUUCA 2337477.1 UCCUACACG AD- A- 4873 UGUAGGAGAATUCA  829-849 A- 5139 UGAAAAGUGAATUC  827-849 1251293.1 2337476.1 CUUUUCA 2337478.1 UCCUACACG AD- A- 4874 UGUAGGAGAATUCA  829-849 A- 5140 UGAAAAAUGAATUC  827-849 1251294.1 2337479.1 UUUUUCA 2337480.1 UCCUACACG AD- A- 4875 UGUAGGAGAAUUCA  829-849 A- 5141 UGAAAAGUGAAUU  827-849 1251344.1 2337550.1 CUUUUCA 2337551.1 CUCCUACACG AD- A- 4876 UGUAGGAGAAUUCA  829-849 A- 5142 UGAAAAGUGAATUC  827-849 1251300.1 2337481.1 CUUUUCA 2337486.1 UCCUACACG AD- A- 4877 UGUAGGAGAAUUCA  829-849 A- 5143 UGAAAAGUGAATUC  827-849 1251295.1 2337481.1 CUUUUCA 2337478.1 UCCUACACG AD- A- 4878 UGUAGGAGAAUUCA  829-849 A- 5144 UGAAAAGUGAATUC  827-849 1251296.1 2337482.1 CUUUUCA 2337478.1 UCCUACACG AD- A- 4879 GUAGGAGAAUUCAC  830-850 A- 5145 UAGAAAAGUGAAU  828-850 1251350.1 2337559.1 UUUUCUA 2337560.1 UCUCCUACGC AD- A- 4880 GUAGGAGAAUUCAC  830-850 A- 5146 UAGAAAAGUGAAU  828-850 1251351.1 2337561.1 UUUUCUA 2337562.1 UCUCCUACGC AD- A- 4881 UAGGAGAAUUCACU  831-851 A- 5147 UAAGAAAAGUGAA  829-851 1251353.1 2337565.1 UUUCUUA 2337566.1 UUCUCCUACG AD- A- 4882 UAGGAGAAUUCACU  831-851 A- 5148 UAAGAAAAGUGAA  829-851 1251352.1 2337563.1 UUUCUUA 2337564.1 UUCUCCUACG AD- A- 4883 UAGGAGAAUUCACU  831-849 A- 5149 UGAAAAGUGAATUC  829-849 1251298.1 2337485.1 UUUCA 2337484.1 UCCUACG AD- A- 4884 UAGGAGAAUUCACU  831-849 A- 5150 UGAAAAGUGAATUC  829-849 1251297.1 2337483.1 UUUCA 2337484.1 UCCUACG AD- A- 4885 AGGAGAAUUCACUU  832-852 A- 5151 UGAAGAAAAGUGA  830-852 1251354.1 2337567.1 UUCUUCA 2337568.1 AUUCUCCUGC AD- A- 4886 GGAGAAUUCACUUU  833-853 A- 5152 UCGAAGAAAAGUGA  831-853 1251355.1 2337569.1 UCUUCGA 2337570.1 AUUCUCCUG AD- A- 4887 GGAGAAUUCACUUU  833-853 A- 5153 UCGAAGAAAAGTGA  831-853 1251356.1 2337571.1 UCUUCGA 2337572.1 AUUCUCCUG AD- A- 4888 GAGAAUUCACUUUU  834-854 A- 5154 UACGAAGAAAAGUG  832-854 1251357.1 2337573.1 CUUCGUA 2337574.1 AAUUCUCCU AD- A- 4889 CCUGAAGCAUAAAU  1108-1128 A- 5155 UGAAAACAUUUAU 1106-1128 1251358.1 2337575.1 GUUUUCA 2337576.1 GCUUCAGGUU AD- A- 4890 CUGAAGCAUAAAUG 1109-1129 A- 5156 UCGAAAACAUUUAU 1107-1129 1251359.1 2337577.1 UUUUCGA 2337578.1 GCUUCAGGU AD- A- 4891 CUGAAGCATAAAUGU 1109-1129 A- 5157 UCGAAAACAUUUAU 1107-1129 1251360.1 2337579.1 UUUCGA 2337580.1 GCUUCAGGU AD- A- 4892 UGAAGCAUAAAUGU 1153-1173 A- 5158 UUCGAAAACAUTUA 1151-1173 1251361.1 1852317.1 UUUCGAA 2337581.1 UGCUUCAGG AD- A- 4893 GAAGCAUAAAUGUU 1111-1131 A- 5159 UUUCGAAAACATUU 1109-1131 1251363.1 2337584.1 UUCGAAA 2337585.1 AUGCUUCAG AD- A- 4894 GAAGCAUAAAUGUU 1111-1131 A- 5160 UUUCGAAAACAUU 1109-1131 1251362.1 2337582.1 UUCGAAA 2337583.1 UAUGCUUCAG AD- A- 4895 AAGCAUAAAUGUUU 1112-1132 A- 5161 UUUUCGAAAACAU 1110-1132 1251364.1 1812604.1 UCGAAAA 2337586.1 UUAUGCUUCG AD- A- 4896 AGCAUAAAUGUUUU 1113-1133 A- 5162 UAUUTCGAAAACAU 1111-1133 1251372.1 2337591.1 CGAAAUA 2337598.1 UUAUGCUUC AD- A- 4897 AGCAUAAAUGUUUU 1113-1133 A- 5163 UAUUUCGAAAACAU 1111-1133 1251366.1 2337589.1 CGAAAUA 1523300.1 UUAUGCUUC AD- A- 4898 AGCAUAAAUGUUUU 1113-1133 A- 5164 UAUUTCGAAAACAU 1111-1133 1251367.1 2337589.1 CGAAAUA 2337590.1 UUAUGCUUC AD- A- 4899 AGCAUAAAUGUUUU 1113-1133 A- 5165 UAUUUCGAAAACAU 1111-1133 795634.4 1523299.1 CGAAAUA 1523300.1 UUAUGCUUC AD- A- 4900 AGCAUAAAUGUUUU 1113-1133 A- 5166 UAUUTCAAAAACAU 1111-1133 1251369.1 2337593.1 UGAAAUA 2337594.1 UUAUGCUUC AD- A- 4901 AGCAUAAAUGUUUU 1113-1133 A- 5167 UAUUTCGAAAACAU 1111-1133 1251368.1 2337591.1 CGAAAUA 2337592.1 UUAUGCUUC AD- A- 4902 AGCAUAAAUGUUUU 1113-1133 A- 5168 UAUUTCGAAAACAU 1111-1133 1251373.1 2337591.1 CGAAAUA 2337599.1 UUAUGCUCC AD- A- 4903 AGCAUAAAUGUUUU 1113-1133 A- 5169 UAUUTCGAAAACAU 1111-1133 1251365.1 2337587.1 CGAAAUA 2337588.1 UUAUGCUUC AD- A- 4904 AGCAUAAAUGUUUU 1113-1133 A- 5170 UAUUTCGAAAACAU 1111-1133 1251370.1 2337591.1 CGAAAUA 2337595.1 UUAUGCUCC AD- A- 4905 GCAUAAAUGUUUUC 1114-1134 A- 5171 UAAUTUCGAAAACA 1112-1134 1251374.1 2337600.1 GAAAUUA 2337601.1 UUUAUGCUU AD- A- 4906 CAUAAAUGUUUUCG 1115-1135 A- 5172 UGAATUTCGAAAAC 1113-1135 1251375.1 2337602.1 AAAUUCA 2337603.1 AUUUAUGCU AD- A- 4907 CAUAAAUGUUUUCG 1115-1133 A- 5173 UAUUTCGAAAACAU 1113-1133 1251371.1 2337596.1 AAAUA 2337597.1 UUAUGCU AD- A- 4908 AUAAAUGUUUUCGA 1116-1136 A- 5174 UUGAAUTUCGAAAA 1114-1136 1251376.1 2337604.1 AAUUCAA 2337605.1 CAUUUAUGC AD- A- 4909 AUAAAUGUUUUCGA 1116-1136 A- 5175 UUGAAUTUCGAAAA 1114-1136 1251377.1 2337604.1 AAUUCAA 2337606.1 CAUUUAUGU AD- A- 4910 UAAAUGUUUUCGAA 1117-1137 A- 5176 UGUGAATUUCGAAA 1115-1137 1251378.1 2337607.1 AUUCACA 2337608.1 ACAUUUAUG AD- A- 4911 AAAUGUUUUCGAAA 1118-1138 A- 5177 UAGUGAAUUUCGA 1116-1138 1251379.1 2337609.1 UUCACUA 2337610.1 AAACAUUUGU AD- A- 4912 UACAUGAUCUUCUU 1430-1450 A- 5178 UACGACAAAGAAGA 1428-1450 1251380.1 2337611.1 UGUCGUA 2337612.1 UCAUGUAGG AD- A- 4913 UACAUGAUCUUCUU 1430-1450 A- 5179 UACGACAAAGAAGA 1428-1450 1251381.1 2337613.1 UGUCGUA 2337614.1 UCAUGUACC AD- A- 4914 ACAUGAUCUUCUUU 1431-1451 A- 5180 UUACGACAAAGAAG 1429-1451 1251382.1 2337615.1 GUCGUAA 2337616.1 AUCAUGUGG AD- A- 4915 CAUGAUCUUCUUUG 1432-1452 A- 5181 UCUACGACAAAGAA 1430-1452 1251384.1 1523843.1 UCGUAGA 2337457.1 GAUCAUGUG AD- A- 4916 CAUGAUCUUCUUUG 1432-1452 A- 5182 UCUACGACAAAGAA 1430-1452 1251274.2 2337449.1 UCGUAGA 2337457.1 GAUCAUGUG AD- A- 4917 CAUGAUCUTCTUUGU 1432-1452 A- 5183 UCUACGACAAAGAA 1430-1452 961188.3 1812612.1 CGUAGA 1812613.1 GAUCAUGUA AD- A- 4918 CAUGAUCUUCUUUG 1432-1452 A- 5184 UCUACGACAAAGAA 1430-1452 1251383.1 1523843.1 UCGUAGA 2337617.1 GAUCAUGUG AD- A- 4919 CAUGAUCUUCUUUG 1432-1452 A- 5185 UCUACGACAAAGAA 1430-1452 1251269.1 2337449.1 UCGUAGA 2337451.1 GAUCAUGUG AD- A- 4920 CAUGAUCUUCUUUG 1432-1452 A- 5186 UCUACGACAAAGAA 1430-1452 1251270.1 2337449.1 UCGUAGA 2337452.1 GAUCAUGCC AD- A- 4921 CAUGAUCUUCUUUG 1432-1452 A- 5187 UCUACGACAAAGAA 1430-1452 1251268.1 2337449.1 UCGUAGA 2337450.1 GAUCAUGUG AD- A- 4922 CAUGAUCUUCUUUG 1432-1452 A- 5188 UCUACGACAAAGAA 1430-1452 1251274.1 2337449.1 UCGUAGA 2337457.1 GAUCAUGUG AD- A- 4923 CAUGAUCUUCUUUG 1432-1452 A- 5189 UCUACGACAAAGAA 1430-1452 1251271.1 2337449.1 UCGUAGA 2337453.1 GAUCAUGCC AD- A- 4924 CAUGAUCUUCUUUG 1432-1452 A- 5190 UCUACGACAAAGAA 1430-1452 1251275.2 2337449.1 UCGUAGA 2337458.1 GAUCAUGUG AD- A- 4925 CAUGAUCUUCUUUG 1432-1452 A- 5191 UCUACGACAAAGAA 1430-1452 1251275.1 2337449.1 UCGUAGA 2337458.1 GAUCAUGUG AD- A- 4926 AUGAUCUUCUUUGU 1433-1453 A- 5192 UACUACGACAAAGA 1431-1453 1251385.1 1523845.1 CGUAGUA 2337618.1 AGAUCAUGU AD- A- 4927 UGAUCUUCUUUGUC 1434-1452 A- 5193 UCUACGACAAAGAA 1432-1452 1251272.1 2337454.1 GUAGA 2337455.1 GAUCAUG AD- A- 4928 UGAUCUUCUUUGUC 1434-1454 A- 5194 UCACTACGACAAAG 1432-1454 1251386.1 1523847.1 GUAGUGA 2337619.1 AAGAUCAUG AD- A- 4929 UGAUCUUCUUUGUC 1434-1452 A- 5195 UCUACGACAAAGAA 1432-1452 1251273.1 2337454.1 GUAGA 2337456.1 GAUCAUG AD- A- 4930 GAUCUUCUUUGUCG 1435-1455 A- 5196 UUCACUACGACAAA 1433-1455 1251390.1 2337622.1 UAGUGAA 2337624.1 GAAGAUCGU AD- A- 4931 GAUCUUCUUUGUCG 1435-1455 A- 5197 UUCACUACGACAAA 1433-1455 1251398.1 2337622.1 UAGUGAA 2337630.1 GAAGAUCGU AD- A- 4932 GAUCUUCUUUGUCG 1435-1455 A- 5198 UUCACUACGACAAA 1433-1455 1251396.1 2337629.1 UAGUGAA 2337621.1 GAAGAUCGU AD- A- 4933 GAUCUUCUUUGUCG 1435-1455 A- 5199 UUCACUACGACAAA 1433-1455 1251399.1 2337628.1 UAGUGAA 2337630.1 GAAGAUCGU AD- A- 4934 GAUCUUCUUUGUCG 1435-1455 A- 5200 UUCACUACGACAAA 1433-1455 795913.3 1523849.1 UAGUGAA 1523850.1 GAAGAUCAU AD- A- 4935 GAUCUUCUUUGUCG 1435-1455 A- 5201 UUCACUACGACAAA 1433-1455 1251400.1 2337629.1 UAGUGAA 2337631.1 GAAGAUCGU AD- A- 4936 GAUCUUCUUUGUCG 1435-1455 A- 5202 UUCACUACGACAAA 1433-1455 1251388.1 1523849.1 UAGUGAA 2337621.1 GAAGAUCGU AD- A- 4937 GAUCUUCUTUGUCG 1435-1455 A- 5203 UUCACUACGACAAA 1433-1455 1251397.1 1812618.1 UAGUGAA 2337624.1 GAAGAUCGU AD- A- 4938 GAUCUUCUUUGUCG 1435-1455 A- 5204 UUCACUACGACAAA 1433-1455 1251395.1 2337628.1 UAGUGAA 2337624.1 GAAGAUCGU AD- A- 4939 GAUCUUCUUUGUCG 1435-1455 A- 5205 UUCACUACGACAAA 1433-1455 1251387.1 1523849.1 UAGUGAA 2337620.1 GAAGAUCGU AD- A- 4940 GAUCUUCUUUGUCG 1435-1455 A- 5206 UUCACUACGACAAA 1433-1455 1251389.1 2337622.1 UAGUGAA 2337623.1 GAAGAUCGU AD- A- 4941 GAUCUUCUUUGUCG 1435-1455 A- 5207 UUCACUACGACAAA 1433-1455 1251393.1 2337628.1 UAGUGAA 2337623.1 GAAGAUCGU AD- A- 4942 GAUCUUCUUUGUCG 1435-1455 A- 5208 UUCACUACGACAAA 1433-1455 1251394.1 2337629.1 UAGUGAA 2337620.1 GAAGAUCGU AD- A- 4943 AUCUUCUUUGUCGU 1436-1456 A- 5209 UAUCACTACGACAA 1434-1456 1251401.1 2337632.1 AGUGAUA 2337633.1 AGAAGAUCG AD- A- 4944 UCUUCUUUGUCGUA 1437-1455 A- 5210 UUCACUACGACAAA 1435-1455 1251391.1 2337625.1 GUGAA 2337626.1 GAAGAUC AD- A- 4945 UCUUCUUUGUCGUA 1437-1455 A- 5211 UUCACUACGACAAA 1435-1455 1251392.1 2337625.1 GUGAA 2337627.1 GAAGAUC AD- A- 4946 UCUUCUUUGUCGUA 1437-1457 A- 5212 UAAUCACUACGACA 1435-1457 1251402.1 2337634.1 GUGAUUA 2337635.1 AAGAAGAUC AD- A- 4947 CUUCUUUGUCGUAG 1438-1458 A- 5213 UAAATCACUACGAC 1436-1458 1251403.1 2337636.1 UGAUUUA 2337637.1 AAAGAAGGU AD- A- 4948 UUCUUUGUCGUAGU 1439-1459 A- 5214 UAAAAUCACUACGA 1437-1459 1251404.1 2337638.1 GAUUUUA 2337639.1 CAAAGAAGG AD- A- 4949 UCUUUGUCGUAGUG 1440-1460 A- 5215 UGAAAATCACUACG 1438-1460 1251405.1 2337640.1 AUUUUCA 2337641.1 ACAAAGAGG AD- A- 4950 AUCCUUUUGUAGAU 2526-2546 A- 5216 UUGCAAGAUCUACA 2524-2546 1251406.1 2337642.1 CUUGCAA 2337643.1 AAAGGAUCC AD- A- 4951 UCCUUUUGUAGAUC 2527-2547 A- 5217 UUUGCAAGAUCTAC 2525-2547 1251407.1 2337644.1 UUGCAAA 2337645.1 AAAAGGAUC AD- A- 4952 CCUUUUGUAGAUCU 2538-2558 A- 5218 UAUUGCAAGAUCU 2536-2558 1251408.1 1854629.1 UGCAAUA 2337646.1 ACAAAAGGGU AD- A- 4953 CUUUUGUAGAUCUU 2529-2549 A- 5219 UAAUTGCAAGAUCU 2527-2549 1251409.1 2337647.1 GCAAUUA 2337648.1 ACAAAAGGG AD- A- 4954 UUUUGUAGAUCUUG 2530-2550 A- 5220 UUAATUGCAAGAUC 1251411.1 2337650.1 CAAUUAA 2337651.1 UACAAAGCC AD- A- 4955 UUUUGUAGAUCUUG 2530-2550 A- 5221 UUAATUGCAAGAUC 2528-2550 1251410.1 1525635.1 CAAUUAA 2337649.1 UACAAAAGG AD- A- 4956 UUUGUAGAUCUUGC 2531-2551 A- 5222 UGUAAUUGCAAGA 2529-2551 1251412.1 2337652.1 AAUUACA 2337653.1 UCUACAAAGG AD- A- 4957 UUUGUAGAUCUUGC 2531-2551 A- 5223 UGUAAUUGCAAGA 2529-2551 796825.3 1525636.1 AAUUACA 1257916.1 UCUACAAAAG AD- A- 4958 UUUGUAGAUCUUGC 2531-2551 A- 5224 UGUAAUTGCAAGAU 2529-2551 1251413.1 2337652.1 AAUUACA 2337654.1 CUACAAAGG AD- A- 4959 UUUGUAGAUCUUGC 2531-2551 A- 5225 UGUAAUUGCAAGA 2529-2551 1251414.1 2337652.1 AAUUACA 2337655.1 UCUACAAAGG AD- A- 4960 UUUGUAGAUCUUGC 2531-2551 A- 5226 UGUAAUTGCAAGAU 2529-2551 1251415.1 2337652.1 AAUUACA 2337656.1 CUACAAAGG AD- A- 4961 UUUGUAGAUCUUGC 2531-2551 A- 5227 UGUAAUUGCAAGA 2529-2551 1251416.1 2337652.1 AAUUACA 2337657.1 UCUACAAAGG AD- A- 4962 UUGUAGAUCUUGCA 2532-2552 A- 5228 UGGUAAUUGCAAG 2530-2552 1251417.1 2337658.1 AUUACCA 2337659.1 AUCUACAAGG AD- A- 4963 UGUAGAUCUUGCAA 2533-2553 A- 5229 UUGGTAAUUGCAAG 2531-2553 1251418.1 2337660.1 UUACCAA 2337661.1 AUCUACAGG AD- A- 4964 GUAGAUCUUGCAAU 2534-2554 A- 5230 UAUGGUAAUUGCA 2532-2554 1251419.1 2337662.1 UACCAUA 2337663.1 AGAUCUACGG AD- A- 4965 UAGAUCUUGCAAUU 2535-2555 A- 5231 UAAUGGTAAUUGCA 2533-2555 1251420.1 2337664.1 ACCAUUA 2337665.1 AGAUCUACG AD- A- 4966 AGAUCUUGCAAUUA 2536-2556 A- 5232 UAAATGGUAAUUGC 2534-2556 1251421.1 1525641.1 CCAUUUA 2337666.1 AAGAUCUGC AD- A- 4967 AGAUCUUGCAAUUA 2536-2556 A- 5233 UAAATGGUAAUTGC 2534-2556 1251422.1 2337667.1 CCAUUUA 2337668.1 AAGAUCUGC AD- A- 4968 UAAAUUAUGUGAAA 3304-3324 A- 5234 UGGUTUGUUUCAC 3302-3324 1251423.1 1856083.1 CAAACCA 2337669.1 AUAAUUUAUU AD- A- 4969 AAUUAUGUGAAACA 3306-3326 A- 5235 UAAGGUUUGUUTC 3304-3326 1251425.1 1856087.1 AACCUUA 2337672.1 ACAUAAUUUG AD- A- 4970 AUUAUGUGAAACAA 3297-3317 A- 5236 UUAAGGTUUGUTUC 3295-3317 1251427.1 2337675.1 ACCUUAA 2337676.1 ACAUAAUUU AD- A- 4971 AUUAUGUGAAACAA 3297-3317 A- 5237 UUAAGGTUUGUUU 3295-3317 1251426.1 2337673.1 ACCUUAA 2337674.1 CACAUAAUUU AD- A- 4972 UUAUGUGAAACAAA 3298-3318 A- 5238 UGUAAGGUUUGUU 3296-3318 1251428.1 2337677.1 CCUUACA 2337678.1 UCACAUAAUU AD- A- 4973 UAUGUGAAACAAACC 3299-3319 A- 5239 UCGUAAGGUUUGU 3297-3319 797564.4 1527042.1 UUACGA 1527043.1 UUCACAUAAU AD- A- 4974 UAUGUGAAACAAACC 3299-3319 A- 5240 UCGUAAGGUUUGU 3297-3319 1251434.1 2337679.1 UUACGA 2337687.1 UUCACAUAGU AD- A- 4975 UAUGUGAAACAAAC 3299-3319 A- 5241 UCGUAAAGUUUGU 3297-3319 1251431.1 2337681.1 UUUACGA 2337682.1 UUCACAUAGU AD- A- 4976 UAUGUGAAACAAACC 3299-3319 A- 5242 UCGUAAGGUUUGU 3297-3319 1251433.1 2337685.1 UUACGA 2337686.1 UUCACAUAGU AD- A- 4977 UAUGUGAAACAAACC 3299-3319 A- 5243 UCGUAAGGUUUGU 3297-3319 1251430.1 2337679.1 UUACGA 2337680.1 UUCACAUAGU AD- A- 4978 UAUGUGAAACAAACC 3299-3319 A- 5244 UCGUAAGGUUUGU 3297-3319 1251429.1 2337679.1 UUACGA 1527043.1 UUCACAUAAU AD- A- 4979 UAUGUGAAACAAACC 3299-3319 A- 5245 UCGUAAGGUUUGU 3297-3319 1251435.1 2337685.1 UUACGA 2337688.1 UUCACAUAGU AD- A- 4980 AUGUGAAACAAACCU 3300-3320 A- 5246 UACGTAAGGUUUG 3298-3320 1251438.1 2337689.1 UACGUA 2337691.1 UUUCACAUGG AD- A- 4981 AUGUGAAACAAACCU 3300-3320 A- 5247 UACGUAAGGUUUG 3298-3320 1251436.1 2337689.1 UACGUA 1527045.1 UUUCACAUAA AD- A- 4982 AUGUGAAACAAACCU 3300-3320 A- 5248 UACGTAAGGUUUG 3298-3320 1251437.1 2337689.1 UACGUA 2337690.1 UUUCACAUGG AD- A- 4983 AUGUGAAACAAACCU 3300-3320 A- 5249 UACGUAAGGUUUG 3298-3320 797565.4 1527044.1 UACGUA 1527045.1 UUUCACAUAA AD- A- 4984 AUGUGAAACAAACCU 3300-3320 A- 5250 UACGTAAGGUUUG 3298-3320 1251443.1 2337689.1 UACGUA 2337698.1 UUUCACAUGG AD- A- 4985 AUGUGAAACAAACCU 3300-3320 A- 5251 UACGTAAGGUUTGU 3298-3320 1251444.1 2337695.1 UACGUA 2337699.1 UUCACAUGG AD- A- 4986 AUGUGAAACAAACCU 3300-3320 A- 5252 UACGTAAGGUUTGU 3298-3320 1251442.1 2337695.1 UACGUA 2337697.1 UUCACAUGG AD- A- 4987 AUGUGAAACAAACCU 3300-3320 A- 5253 UACGTAAGGUUTGU 3298-3320 1251441.1 2337695.1 UACGUA 2337696.1 UUCACAUGG AD- A- 4988 UGUGAAACAAACCU 3301-3321 A- 5254 UCACGUAAGGUTUG 3299-3321 1251445.1 2337700.1 UACGUGA 2337701.1 UUUCACAUG AD- A- 4989 GUGAAACAAACCUUA 3302-3320 A- 5255 UACGTAAGGUUUG 3300-3320 1251439.1 2337692.1 CGUA 2337693.1 UUUCACGU AD- A- 4990 GUGAAACAAACCUUA 3302-3322 A- 5256 UUCACGTAAGGTUU 3300-3322 1251447.1 2337704.1 CGUGAA 2337705.1 GUUUCACGU AD- A- 4991 GUGAAACAAACCUUA 3302-3322 A- 5257 UUCACGTAAGGUUU 3300-3322 1251446.1 2337702.1 CGUGAA 2337703.1 GUUUCACGU AD- A- 4992 UGAAACAAACCUUAC 3303-3323 A- 5258 UUUCACGUAAGGU 3301-3323 1251448.1 2337706.1 GUGAAA 2337707.1 UUGUUUCACA AD- A- 4993 GAAACAAACCUUACG 3304-3324 A- 5259 UAUUCACGUAAGG 3302-3324 1251450.1 2337710.1 UGAAUA 2337711.1 UUUGUUUCAC AD- A- 4994 GAAACAAACCUUACG 3304-3324 A- 5260 UAUUCACGUAAGG 3302-3324 1251449.1 2337708.1 UGAAUA 2337709.1 UUUGUUUCAC AD- A- 4995 AAACAAACCUUACGU 3305-3325 A- 5261 UAAUTCACUGAAGG 1251451.1 2337712.1 GAAUUA 2337713.1 UUUGUUUCG AD- A- 4996 UGUGAUAUAUUUUA 8017-8037 A- 5262 UAUGTUGUAAAAU 8015-8037 1251453.1 2337716.1 CAACAUA 2337717.1 AUAUCACAGU AD- A- 4997 UGUGAUAUAUUUUA 8017-8037 A- 5263 UAUGTUGUAAAAU 8015-8037 1251452.1 2337714.1 CAACAUA 2337715.1 AUAUCACAGU AD- A- 4998 GUGAUAUAUUUUAC 8018-8038 A- 5264 UGAUGUUGUAAAA 8016-8038 1251454.1 2337718.1 AACAUCA 2337719.1 UAUAUCACGG AD- A- 4999 UGAUAUAUUUUACA 8019-8039 A- 5265 UGGATGUUGUAAA 8017-8039 1251455.1 2337720.1 ACAUCCA 2337721.1 AUAUAUCACG AD- A- 5000 GAUAUAUUUUACAA 8020-8040 A- 5266 UCGGAUGUUGUAA 8018-8040 1251456.1 2337722.1 CAUCCGA 2337723.1 AAUAUAUCGC AD- A- 5001 GAUAUAUUUUACAA 8020-8040 A- 5267 UCGGAUGUUGUAA 8018-8040 1251457.1 2337724.1 CAUCCGA 2337725.1 AAUAUAUCGC AD- A- 5002 AUAUAUUUUACAAC 8021-8041 A- 5268 UACGGATGUUGTAA 8019-8041 1251459.1 2337727.1 AUCCGUA 2337728.1 AAUAUAUCG AD- A- 5003 AUAUAUUUUACAAC 8021-8041 A- 5269 UACGGATGUUGUAA 8019-8041 1251458.1 1535069.1 AUCCGUA 2337726.1 AAUAUAUCG AD- A- 5004 UAUAUUUUACAACA 8022-8042 A- 5270 UAACGGAUGUUGU 8020-8042 1251462.1 1535071.1 UCCGUUA 2337731.1 AAAAUAUACC AD- A- 5005 UAUAUUUUACAACA 8022-8042 A- 5271 UAACGGAUGUUGU 8020-8042 1251461.1 1535071.1 UCCGUUA 2337730.1 AAAAUAUAUC AD- A- 5006 UAUAUUUUACAACA 8022-8042 A- 5272 UAACGGAUGUUGU 8020-8042 1251468.1 1535071.1 UCCGUUA 2337738.1 AAAAUAUACC AD- A- 5007 UAUAUUUUACAACA 8022-8042 A- 5273 UAACGGAUGUUGU 8020-8042 1251463.1 1535071.1 UCCGUUA 2337732.1 AAAAUAUACC AD- A- 5008 UAUAUUUUACAACA 8022-8042 A- 5274 UAACGGAUGUUGU 8020-8042 1251460.1 1535071.1 UCCGUUA 2337729.1 AAAAUAUAUC AD- A- 5009 UAUAUUUUACAACA 8032-8052 A- 5275 UAACGGAUGUUGU 8030-8052 1251469.1 1864159.1 UCCGUUA 2337739.1 AAAAUAUAUC AD- A- 5010 UAUAUUUUACAACA 8022-8042 A- 5276 UAACGGAUGUUGU 8020-8042 801647.3 1535071.1 UCCGUUA 1535072.1 AAAAUAUAUC AD- A- 5011 UAUAUUUUACAACA 8032-8052 A- 5277 UAACGGAUGUUGU 8030-8052 1251467.1 1864159.1 UCCGUUA 2337737.1 AAAAUAUAUC AD- A- 5012 UAUAUUUUACAACA 8022-8042 A- 5278 UAACGGAUGUUGU 8020-8042 1251466.1 2337736.1 UCCGUUA 2337737.1 AAAAUAUAUC AD- A- 5013 AUAUUUUACAACAU 8023-8043 A- 5279 UUAACGGAUGUUG 8021-8043 1251470.1 1535073.1 CCGUUAA 2337740.1 UAAAAUAUGU AD- A- 5014 AUAUUUUACAACAU 8023-8043 A- 5280 UUAACGGAUGUTG 8021-8043 1251471.1 2337741.1 CCGUUAA 2337742.1 UAAAAUAUGU AD- A- 5015 UAUUUUACAACAUC 8024-8042 A- 5281 UAACGGAUGUUGU 8022-8042 1251465.1 2337733.1 CGUUA 2337735.1 AAAAUAUG AD- A- 5016 UAUUUUACAACAUC 8024-8044 A- 5282 UAUAACGGAUGTUG 8022-8044 1251472.1 2337743.1 CGUUAUA 2337744.1 UAAAAUAUG AD- A- 5017 UAUUUUACAACAUC 8024-8042 A- 5283 UAACGGAUGUUGU 8022-8042 1251464.1 2337733.1 CGUUA 2337734.1 AAAAUAUG AD- A- 5018 AUUUUACAACAUCCG 8025-8045 A- 5284 UAAUAACGGAUGU 8023-8045 1251473.1 2337745.1 UUAUUA 2337746.1 UGUAAAAUGU AD- A- 5019 AUUUUACAACAUCCG 8025-8045 A- 5285 UAAUAACGGAUGU 8023-8045 1251474.1 2337747.1 UUAUUA 2337748.1 UGUAAAAUGU AD- A- 5020 UUUUACAACAUCCG 8026-8046 A- 5286 UUAATAACGGATGU 8024-8046 1251475.1 2337749.1 UUAUUAA 2337750.1 UGUAAAAUG AD- A- 5021 UUUACAACAUCCGU 8027-8047 A- 5287 UGUAAUAACGGAU 8025-8047 1251476.1 2337751.1 UAUUACA 2337752.1 GUUGUAAAGU AD- A- 5022 CAACACAAUUUCUUC 8498-8518 A- 5288 UGCUAAGAAGAAAU 8496-8518 1251279.1 2337459.1 UUAGCA 2337464.1 UGUGUUGUU AD- A- 5023 CAACACAAUUUCUUC 8498-8518 A- 5289 UGCUAAGAAGAAAU 8496-8518 1251276.1 2337459.1 UUAGCA 2337460.1 UGUGUUGUU AD- A- 5024 CAACACAAUUUCUUC 8498-8518 A- 5290 UGCUAAGAAGAAAU 8496-8518 1251280.1 2337459.1 UUAGCA 2337465.1 UGUGUUGCC AD- A- 5025 CAACACAAUUUCUUC 8498-8518 A- 5291 UGCUAAGAAGAAAU 8496-8518 1251277.1 2337459.1 UUAGCA 2337461.1 UGUGUUGCC AD- A- 5026 CAACACAATUTCUUC 8498-8518 A- 5292 UGCUAAGAAGAAAT 8496-8518 961334.3 1812904.1 UUAGCA 1812905.1 UGUGUUGUU AD- A- 5027 ACACAAUUUCUUCU 8500-8518 A- 5293 UGCUAAGAAGAAAU 8498-8518 1251278.1 2337462.1 UAGCA 2337463.1 UGUGUUG AD- A- 5028 GGCUUCAAGUGUUC 9109-9129 A- 5294 UCAGTAGGAACACU 9107-9129 1251477.1 1865763.1 CUACUGA 2337753.1 UGAAGCCGG AD- A- 5029 GCUUCAAGUGUUCC 9100-9120 A- 5295 UACAGUAGGAACAC 9098-9120 1251478.1 2337754.1 UACUGUA 2337755.1 UUGAAGCCG AD- A- 5030 CUUCAAGUGUUCCU 9101-9121 A- 5296 UGACAGTAGGAACA 9099-9121 1251479.1 2337756.1 ACUGUCA 2337757.1 CUUGAAGCC AD- A- 5031 UUCAAGUGUUCCUA 9102-9122 A- 5297 UUGACAGUAGGAAC 9100-9122 1251481.1 2337758.1 CUGUCAA 2337760.1 ACUUGAAGC AD- A- 5032 UUCAAGUGUUCCUA 9102-9122 A- 5298 UUGACAGUAGGAAC 9100-9122 1251480.1 2337758.1 CUGUCAA 2337759.1 ACUUGAAGC AD- A- 5033 UCAAGUGUUCCUAC 9103-9123 A- 5299 UAUGACAGUAGGA 9101-9123 1251482.1 2337761.1 UGUCAUA 2337762.1 ACACUUGAGG AD- A- 5034 UCAAGUGUUCCUAC 9103-9123 A- 5300 UAUGACAGUAGGA 9101-9123 1251483.1 2337761.1 UGUCAUA 2337763.1 ACACUUGAGG AD- A- 5035 CAAGUGUUCCUACU 9104-9124 A- 5301 UCAUGACAGUAGGA 9102-9124 1251492.1 2337764.1 GUCAUGA 2337773.1 ACACUUGCC AD- A- 5036 CAAGUGUUCCUACU 9104-9124 A- 5302 UCAUGACAGUAGGA 9102-9124 1251485.1 2337764.1 GUCAUGA 2337766.1 ACACUUGGG AD- A- 5037 CAAGUGUUCCUACU 9104-9124 A- 5303 UCAUGACAGUAGGA 9102-9124 802471.4 1536717.1 GUCAUGA 1536718.1 ACACUUGAA AD- A- 5038 CAAGUGUUCCUACU 9104-9124 A- 5304 UCAUGACAGUAGGA 9102-9124 1251486.1 2337764.1 GUCAUGA 1536718.1 ACACUUGAA AD- A- 5039 CAAGUGUUCCUACU 9104-9124 A- 5305 UCAUGACAGUAGGA 9102-9124 1251484.1 2337764.1 GUCAUGA 2337765.1 ACACUUGGG AD- A- 5040 CAAGUGUUCCUACU 9104-9124 A- 5306 UCAUGACAGUAGGA 9102-9124 1251491.1 2337764.1 GUCAUGA 2337772.1 ACACUUGGG AD- A- 5041 CAAGUGUUCCUACU 9104-9124 A- 5307 UCAUGACAGUAGGA 9102-9124 1251487.1 2337764.1 GUCAUGA 2337767.1 ACACUUGGG AD- A- 5042 CAAGUGUUCCUACU 9104-9124 A- 5308 UCAUGACAGUAGGA 9102-9124 1251488.1 2337764.1 GUCAUGA 2337768.1 ACACUUGCC AD- A- 5043 CAAGUGUUCCUACU 9104-9124 A- 5309 UCAUGACAGUAGGA 9102-9124 1251490.1 2337764.1 GUCAUGA 2337771.1 ACACUUGGG AD- A- 5044 AAGUGUUCCUACUG 9105-9125 A- 5310 UUCATGACAGUAGG 9103-9125 1251494.1 2337775.1 UCAUGAA 2337776.1 AACACUUGG AD- A- 5045 AGUGUUCCUACUGU 9106-9124 A- 5311 UCAUGACAGUAGGA 9104-9124 1251493.1 2337769.1 CAUGA 2337774.1 ACACUUG AD- A- 5046 AGUGUUCCUACUGU 9106-9124 A- 5312 UCAUGACAGUAGGA 9104-9124 1251489.1 2337769.1 CAUGA 2337770.1 ACACUUG AD- A- 5047 AGUGUUCCUACUGU 9106-9126 A- 5313 UGUCAUGACAGTAG 9104-9126 1251495.1 2337777.1 CAUGACA 2337778.1 GAACACUUG AD- A- 5048 GUGUUCCUACUGUC 9107-9127 A- 5314 UGGUCATGACAGUA 9105-9127 1251496.1 2337779.1 AUGACCA 2337780.1 GGAACACUU AD- A- 5049 UGUUCCUACUGUCA 9108-9128 A- 5315 UAGGTCAUGACAGU 9106-9128 1251497.1 2337781.1 UGACCUA 2337782.1 AGGAACACU AD- A- 5050 GUUCCUACUGUCAU 9109-9129 A- 5316 UCAGGUCAUGACAG 9107-9129 1251498.1 2337783.1 GACCUGA 2337784.1 UAGGAACGC AD- A- 5051 UUGAUAGUUACCUA 9225-9245 A- 5317 UGCAAACUAGGUAA 9223-9245 802552.3 1536877.1 GUUUGCA 1536878.1 CUAUCAAAA AD- A- 5052 UUGAUAGUTACCUA 9225-9245 A- 5318 UGCAAACUAGGTAA 9223-9245 1251267.1 2337439.1 GUUUGCA 2337448.1 CUAUCAAGG AD- A- 5053 UUGAUAGUTACCUA 9225-9245 A- 5319 UGCAAACUAGGTAA 9223-9245 1251260.1 2337439.1 GUUUGCA 2337438.1 CUAUCAAGG AD- A- 5054 UUGAUAGUUACCUA 9225-9245 A- 5320 UGCAAACUAGGUAA 9223-9245 1251256.1 2337433.1 GUUUGCA 1536878.1 CUAUCAAAA AD- A- 5055 UUGAUAGUUACCUA 9225-9245 A- 5321 UGCAAACUAGGUAA 9223-9245 1251265.1 2337436.1 GUUUGCA 2337447.1 CUAUCAAGG AD- A- 5056 UUGAUAGUUACCUA 9225-9245 A- 5322 UGCAAACUAGGUAA 9223-9245 1251257.1 2337434.1 GUUUGCA 2337435.1 CUAUCAAGG AD- A- 5057 UUGAUAGUUACCUA 9225-9245 A- 5323 UGCAAACUAGGTAA 9223-9245 1251266.1 2337437.1 GUUUGCA 2337448.1 CUAUCAAGG AD- A- 5058 UUGAUAGUUACCUA 9225-9245 A- 5324 UGCAAATUAGGUAA 1251264.1 2337445.1 AUUUGCA 2337446.1 CUAUCAAGG AD- A- 5059 UUGAUAGUUACCUA 9225-9245 A- 5325 UGCAAACUAGGTAA 9223-9245 1251259.1 2337437.1 GUUUGCA 2337438.1 CUAUCAAGG AD- A- 5060 UUGAUAGUUACCUA 9225-9245 A- 5326 UGCAAACUAGGUAA 9223-9245 1251258.1 2337436.1 GUUUGCA 2337435.1 CUAUCAAGG AD- A- 5061 GAUAGUTACCUAGU 9227-9245 A- 5327 UGCAAACUAGGTAA 9225-9245 1251263.1 2337444.1 UUGCA 2337443.1 CUAUCGG AD- A- 5062 GAUAGUUACCUAGU 9227-9245 A- 5328 UGCAAACUAGGTAA 9225-9245 1251262.1 2337442.1 UUGCA 2337443.1 CUAUCGG AD- A- 5063 GAUAGUUACCUAGU 9227-9245 A- 5329 UGCAAACUAGGUAA 9225-9245 1251261.1 2337440.1 UUGCA 2337441.1 CUAUCGG

TABLE 14A Exemplary Human SCN9A siRNA Modified Single Strands and Duplex Sequences Column 1 indicates duplex name and the number following the decimal point in a duplex name merely refers to a batch production number. Column 2 indicates the name of the sense sequence. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the modified sequence of a sense strand suitable for use in a duplex described herein. Column 5 indicates the antisense sequence name. Column 6 indicates the sequence ID for the sequence of column 7. Column 7 provides the sequence of a modified antisense strand suitable for use in a duplex described herein, e.g., a duplex comprising the sense sequence in the same row of the table. Column 8 indicates the position in the target mRNA (NM_001365536.1) that is complementary to the antisense strand of Column 7. Column 9 indicated the sequence ID for the sequence of column 8. Sense Seq ID Antisense Seq ID mRNA target SEQ ID NO: Duplex sequence NO: Sense sequence sequence NO: Antisense sequence sequence in (mRNA Name name (sense) (5′-3′) name (antisense) (5′-3′) NM_001365536.1 target) AD- A- 5330 usgsucg(Ahd)GfuAf A- 5346 VPusCfsaguAfaAfAfgu AAUGUCGAGUACACU 5362 795305.2 1522697.1 CfAfcuuuuacugaL96 1522698.1 guAfcUfcgacasusu UUUACUGG AD- A- 5331 usgsucgaguAfCfAfc A- 5347 VPusCfsagdTadAaagu AAUGUCGAGUACACU 5363 1251249.1 2337423.1 uuu(Uhd)acugaL96 2337424.1 guAfcUfcgacasusu UUUACUGG AD- A- 5332 uscsgaguAfCfAfcuu A- 5348 VPusCfsagdTadAaagu UGUCGAGUACACUUU 5364 1251251.1 2337426.1 u(Uhd)acugaL96 2337427.1 guAfcUfcgascsg UACUGG AD- A- 5333 usgsuag(Ghd)agdAa A- 5349 VPusdGsaadAadGuga UGUGUAGGAGAAUUC 5365 1010663.2 1851796.1 dTucacuuuucaL96 1875201.1 adTudCudCcuacascsa ACUUUUCU AD- A- 5334 usgsuaggagdAaUfU A- 5350 VPudGaadAa(G2p)ug UGUGUAGGAGAAUUC 5366 1251301.1 2337482.1 fcac(Uhd)uuucaL96 2337486.1 aadTudCudCcuacascs ACUUUUCU g AD- A- 5335 asasggg(Ahd)aadAc A- 5351 VPusdAscgdGadAgau CAAAGGGAAAACAAU 5367 961179.3 1812594.1 dAaucuuccguaL96 1812595.1 udGudTudTcccuusus CUUCCGUU g AD- A- 5336 asasgggaaaAfCfAfa A- 5352 VPudAcgdGa(A2p)ga CAAAGGGAAAACAAU 5368 1251317.1 2337506.1 ucu(Uhd)ccguaL96 2337508.1 uudGuUfudTcccuusus CUUCCGUU g AD- A- 5337 asgsggaaAfaCfAfAfu A- 5353 VPusAfsacdGgdAagau AAAGGGAAAACAAUC 5369 1251318.1 2337509.1 cuu(Chd)cguuaL96 2337510.1 ugUfuUfucccususu UUCCGUUU AD- A- 5338 gsasaaa(Chd)aaUfCf A- 5354 VPuUfgadAa(C2p)gga GGGAAAACAAUCUUC 5370 1251323.1 2337519.1 UfuccguuucaaL96 2337520.1 agaUfudGuuuucscsc CGUUUCAA AD- A- 5339 asasaacaauCfUfUfc A- 5355 VPuUfugdAadAcggad GGAAAACAAUCUUCC 5371 1251325.1 2337523.1 cgu(Uhd)ucaaaL96 2337524.1 AgdAuUfguuuuscsc GUUUCAAU AD- A- 5340 asgscau(Ahd)AfaUf A- 5356 VPusAfsuuuCfgAfAfaa GAAGCAUAAAUGUUU 5372 795634.3 1523299.1 GfUfuuucgaaauaL9 1523300.1 caUfuUfaugcususc UCGAAAUU AD- A- 5341 gsasagcauadAaUfgu A- 5357 VPuUfucdGadAaacad CUGAAGCAUAAAUGU 5373 1251363.1 2337584.1 uu(Uhd)cgaaaL96 2337585.1 TuUfaUfgcuucsasg UUUCGAAA AD- A- 5342 asasgca(Uhd)aadAu A- 5358 VPuUfuudCgdAaaacd UGAAGCAUAAAUGUU 5374 1251364.1 1812604.1 dGuuuucgaaaaL96 2337586.1 AuUfudAugcuuscsg UUCGAAAU AD- A- 5343 asgscauaaaUfgUfuu A- 5359 VPudAuudTc(G2p)aaa GAAGCAUAAAUGUUU 5375 1251373.1 2337591.1 u(Chd)gaaauaL96 2337599.1 adCaUfuUfaugcuscsc UCGAAAUU AD- A- 5344 asusgau(Chd)UfuCf A- 5360 VPudAcudAcdGacaad ACAUGAUCUUCUUUG 5376 1251385.1 1523845.1 UfUfugucguaguaL96 2337618.1 AgdAadGaucausgsu UCGUAGUG AD- A- 5345 uscsu(Uhd)CfuUfud A- 5361 VPusUfscadCu(Agn)cg GAUCUUCUUUGUCGU 5377 1251391.1 2337625.1 GucguagugaaL96 2337626.1 acdAaAfgAfagasusc AGUGAU

TABLE 14B Exemplary Human SCN9A Unmodified Single Strands and Duplex Sequences. Column 1 indicates duplex name; the number following the decimal point in a duplex name merely refers to a batch production number. Column 2 indicates the sense sequence name. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the unmodified sequence of a sense strand suitable for use in a duplex described herein. Column 5 provides the position in the target mRNA (NM_001365536.1) of the sense strand of Column 4. Column 6 indicates the antisense sequence name. Column 7 indicates the sequence ID for the sequence of column 8. Column 8 provides the sequence of an antisense strand suitable for use in a duplex described herein, without specifying chemical modifications. Column 9 indicates the position in the target mRNA (NM_001365536.1) that is complementary to the antisense strand of Column 8. Sense Seq ID mRNA target Antisense mRNA target Duplex sequence NO: Sense sequence range in sequence Seq ID NO: antisense sequence range in Name name (sense) (5′-3′) NM_001365536.1 name (antisense) (5′-3′) NM_001365536.1 AD- A- 5378 UGUCGAGUACACUU  760-780 A- 5394 UCAGUAAAAGUGU  758-780 795305.2 1522697.1 UUACUGA 1522698.1 ACUCGACAUU AD- A- 5379 UGUCGAGUACACUU  760-780 A- 5395 UCAGTAAAAGUGUA  758-780 1251249.1 2337423.1 UUACUGA 2337424.1 CUCGACAUU AD- A- 5380 UCGAGUACACUUUU  762-780 A- 5396 UCAGTAAAAGUGUA  760-780 1251251.1 2337426.1 ACUGA 2337427.1 CUCGACG AD- A- 5381 UGUAGGAGAATUCA  872-892 A- 5397 UGAAAAGUGAATUC  870-892 1010663.2 1851796.1 CUUUUCA 1875201.1 UCCUACACA AD- A- 5382 UGUAGGAGAAUUCA  829-849 A- 5398 UGAAAAGUGAATUC  827-849 1251301.1 2337482.1 CUUUUCA 2337486.1 UCCUACACG AD- A- 5383 AAGGGAAAACAAUCU  576-596 A- 5399 UACGGAAGAUUGUT  574-596 961179.3 1812594.1 UCCGUA 1812595.1 UTCCCUUUG AD- A- 5384 AAGGGAAAACAAUCU  576-596 A- 5400 UACGGAAGAUUGU  574-596 1251317.1 2337506.1 UCCGUA 2337508.1 UUTCCCUUUG AD- A- 5385 AGGGAAAACAAUCU  577-597 A- 5401 UAACGGAAGAUUG  575-597 1251318.1 2337509.1 UCCGUUA 2337510.1 UUUUCCCUUU AD- A- 5386 GAAAACAAUCUUCCG  580-600 A- 5402 UUGAAACGGAAGA  578-600 1251323.1 2337519.1 UUUCAA 2337520.1 UUGUUUUCCC AD- A- 5387 AAAACAAUCUUCCGU  581-601 A- 5403 UUUGAAACGGAAG  579-601 1251325.1 2337523.1 UUCAAA 2337524.1 AUUGUUUUCC AD- A- 5388 AGCAUAAAUGUUUU 1113-1133 A- 5404 UAUUUCGAAAACAU 1111-1133 795634.3 1523299.1 CGAAAUA 1523300.1 UUAUGCUUC AD- A- 5389 GAAGCAUAAAUGUU 1111-1131 A- 5405 UUUCGAAAACATUU 1109-1131 1251363.1 2337584.1 UUCGAAA 2337585.1 AUGCUUCAG AD- A- 5390 AAGCAUAAAUGUUU 1112-1132 A- 5406 UUUUCGAAAACAU 1110-1132 1251364.1 1812604.1 UCGAAAA 2337586.1 UUAUGCUUCG AD- A- 5391 AGCAUAAAUGUUUU 1113-1133 A- 5407 UAUUTCGAAAACAU 1111-1133 1251373.1 2337591.1 CGAAAUA 2337599.1 UUAUGCUCC AD- A- 5392 AUGAUCUUCUUUGU 1433-1453 A- 5408 UACUACGACAAAGA 1431-1453 1251385.1 1523845.1 CGUAGUA 2337618.1 AGAUCAUGU AD- A- 5393 UCUUCUUUGUCGUA 1437-1455 A- 5409 UUCACUACGACAAA 1435-1455 1251391.1 2337625.1 GUGAA 2337626.1 GAAGAUC

TABLE 15A Exemplary Human SCN9A siRNA Modified Single Strands and Duplex Sequences Column 1 indicates duplex name and the number following the decimal point in a duplex name merely refers to a batch production number. Column 2 indicates the name of the sense sequence. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the modified sequence of a sense strand suitable for use in a duplex described herein. Column 5 indicates the antisense sequence name. Column 6 indicates the sequence ID for the sequence of column 7. Column 7 provides the sequence of a modified antisense strand suitable for use in a duplex described herein, e.g., a duplex comprising the sense sequence in the same row of the table. Column 8 indicates the position in the target mRNA (NM_001365536.1) that is complementary to the antisense strand of Column 7. Column 9 indicated the sequence ID for the sequence of column 8. Seq ID Sense Seq ID Antisense NO: mRNA target SEQ ID NO: Duplex sequence NO: Sense sequence sequence (anti Antisense sequence sequence in (mRNA Name name (sense) (5′-3′) name sense) (5′-3′) NM_001365536.1 target) AD- A- 5410 csasagugUfuCfCfUf A- 5426 VPuCfaudGa(C2p)agu UUCAAGUGUUCCUAC 5442 1251492.2 2337764.1 acug(Uhd)caugaL96 2337773.1 aggAfaCfacuugscsc UGUCAUGA AD- A- 5411 csasaca(Chd)aadTu A- 5427 VPusdGscudAadGaag AACAACACAAUUUCU 5443 961334.2 1812904.1 dTcuucuuagcaL96 1812905.1 adAadTudGuguugsus UCUUAGCA U AD- A- 5412 csasaca(Chd)aaUfUf A- 5428 VPudGcudAadGaagad AACAACACAAUUUCU 5444 1251279.2 2337459.1 UfcuucuuagcaL96 2337464.1 AaUfudGuguugsusu UCUUAGCA AD- A- 5413 usgsucgaguAfCfAfc A- 5429 VPusCfsagdTadAaagu AAUGUCGAGUACACU 5445 1251284.2 2337423.1 uuu(Uhd)acugaL96 2337467.1 dGuAfcdTcgacasusu UUUACUGG AD- A- 5414 ususcug(Uhd)guAfg A- 5430 VPusdGsugdAa(U2p) GCUUCUGUGUAGGAG 5446 1251334.2 2337536.1 dGagaauucacaL96 2337538.1 ucucdCuAfcAfcagaas AAUUCACU gsc AD- A- 5415 asusaaa(Uhd)guUfU A- 5431 VPusUfsgadAudTucga GCAUAAAUGUUUUCG 5447 1251377.2 2337604.1 fUfcgaaauucaaL96 2337606.1 aaAfcAfuuuausgsu AAAUUCAC AD- A- 5416 gsasucu(Uhd)CfuUf A- 5432 VPuUfcadCu(A2p)cga AUGAUCUUCUUUGUC 5448 1251398.2 2337622.1 udGucguagugaaL96 2337630.1 cdAaAfgAfagaucsgsu GUAGUGAU AD- A- 5417 gsasucu(Uhd)CfuUf A- 5433 VPuUfcadCu(A2p)cga AUGAUCUUCUUUGUC 5449 1251399.2 2337628.1 udGUfcguagugaaL96 2337630.1 cdAaAfgAfagaucsgsu GUAGUGAU AD- A- 5418 csasuga(Uhd)cudTc A- 5434 VPusdCsuadCgdAcaa UACAUGAUCUUCUUU 5450 961188.2 1812612.1 dTuugucguagaL96 1812613.1 adGadAgdAucaugsus GUCGUAGU a AD- A- 5419 csasuga(Uhd)cuUfC A- 5435 VPuCfuadCgdAcaaad UACAUGAUCUUCUUU 5451 1251274.3 2337449.1 fUfuugucguagaL96 2337457.1 GadAgdAucaugsusg GUCGUAGU AD- A- 5420 ususugu(Ahd)GfaUf A- 5436 VPusGfsuaaUfuGfCfa CUUUUGUAGAUCUU 5452 796825.2 1525636.1 CfUfugcaauuacaL96 1257916.1 agaUfcUfacaaasasg GCAAUUACC AD- A- 5421 ususuug(Uhd)agAfU A- 5437 VPusUfsaadTu(G2p)c 1251411.2 2337650.1 fCfuugcaauuaaL96 2337651.1 aagauCfuAfcaaagscsc AD- A- 5422 gsusaga(Uhd)CfuUf A- 5438 VPudAugdGudAauug UUGUAGAUCUUGCAA 5454 1251419.2 2337662.1 gCfaauuaccauaL96 2337663.1 dCaAfgAfucuacsgsg UUACCAUU AD- A- 5423 usasugu(Ghd)AfaAf A- 5439 VPusCfsguaAfgGfUfu AUUAUGUGAAACAAA 5455 797564.3 1527042.1 CfAfaaccuuacgaL96 1527043.1 uguUfuCfacauasasu CCUUACGU AD- A- 5424 ususaug(Uhd)gaAfA A- 5440 VPudGuadAg(G2p)uu AAUUAUGUGAAACAA 5456 1251428.2 2337677.1 fCfaaaccuuacaL96 2337678.1 uguuUfcAfcauaasusu ACCUUACG AD- A- 5425 usasugugAfaAfCfAf A- 5441 VPuCfgudAa(G2p)guu AUUAUGUGAAACAAA 5457 1251434.2 2337679.1 aacc(Uhd)uacgaL96 2337687.1 uguUfuCfacauasgsu CCUUACGU

TABLE 15B Exemplary Human SCN9A Unmodified Single Strands and Duplex Sequences. Column 1 indicates duplex name; the number following the decimal point in a duplex name merely refers to a batch production number. Column 2 indicates the sense sequence name. Column 3 indicates the sequence ID for the sequence of column 4. Column 4 provides the unmodified sequence of a sense strand suitable for use in a duplex described herein. Column 5 provides the position in the target mRNA (NM_001365536.1) of the sense strand of Column 4. Column 6 indicates the antisense sequence name. Column 7 indicates the sequence ID for the sequence of column 8. Column 8 provides the sequence of an antisense strand suitable for use in a duplex described herein, without specifying chemical modifications. Column 9 indicates the position in the target mRNA (NM_001365536.1) that is complementary to the antisense strand of Column 8. Sense Seq ID mRNA target Antisense mRNA target Duplex sequence NO: Sense sequence range in sequence Seq ID NO: antisense sequence range in Name name (sense) (5′-3′) NM_001365536.1 name (antisense) (5′-3′) NM_001365536.1 AD- A- 5458 CAAGUGUUCCUACU 9104-9124 A- 5474 UCAUGACAGUAGGA 9102-9124 1251492.2 2337764.1 GUCAUGA 2337773.1 ACACUUGCC AD- A- 5459 CAACACAATUTCUUC 8498-8518 A- 5475 UGCUAAGAAGAAAT 8496-8518 961334.2 1812904.1 UUAGCA 1812905.1 UGUGUUGUU AD- A- 5460 CAACACAAUUUCUUC 8498-8518 A- 5476 UGCUAAGAAGAAAU 8496-8518 1251279.2 2337459.1 UUAGCA 2337464.1 UGUGUUGUU AD- A- 5461 UGUCGAGUACACUU  760-780 A- 5477 UCAGTAAAAGUGUA  758-780 1251284.2 2337423.1 UUACUGA 2337467.1 CTCGACAUU AD- A- 5462 UUCUGUGUAGGAGA  824-844 A- 5478 UGUGAAUUCUCCU  822-844 1251334.2 2337536.1 AUUCACA 2337538.1 ACACAGAAGC AD- A- 5463 AUAAAUGUUUUCGA 1116-1136 A- 5479 UUGAAUTUCGAAAA 1114-1136 1251377.2 2337604.1 AAUUCAA 2337606.1 CAUUUAUGU AD- A- 5464 GAUCUUCUUUGUCG 1435-1455 A- 5480 UUCACUACGACAAA 1433-1455 1251398.2 2337622.1 UAGUGAA 2337630.1 GAAGAUCGU AD- A- 5465 GAUCUUCUUUGUCG 1435-1455 A- 5481 UUCACUACGACAAA 1433-1455 1251399.2 2337628.1 UAGUGAA 2337630.1 GAAGAUCGU AD- A- 5466 CAUGAUCUTCTUUGU 1432-1452 A- 5482 UCUACGACAAAGAA 1430-1452 961188.2 1812612.1 CGUAGA 1812613.1 GAUCAUGUA AD- A- 5467 CAUGAUCUUCUUUG 1432-1452 A- 5483 UCUACGACAAAGAA 1430-1452 1251274.3 2337449.1 UCGUAGA 2337457.1 GAUCAUGUG AD- A- 5468 UUUGUAGAUCUUGC 2531-2551 A- 5484 UGUAAUUGCAAGA 2529-2551 796825.2 1525636.1 AAUUACA 1257916.1 UCUACAAAAG AD- A- 5469 UUUUGUAGAUCUUG 2530-2550 A- 5485 UUAATUGCAAGAUC 1251411.2 2337650.1 CAAUUAA 2337651.1 UACAAAGCC AD- A- 5470 GUAGAUCUUGCAAU 2534-2554 A- 5486 UAUGGUAAUUGCA 2532-2554 1251419.2 2337662.1 UACCAUA 2337663.1 AGAUCUACGG AD- A- 5471 UAUGUGAAACAAACC 3299-3319 A- 5487 UCGUAAGGUUUGU 3297-3319 797564.3 1527042.1 UUACGA 1527043.1 UUCACAUAAU AD- A- 5472 UUAUGUGAAACAAA 3298-3318 A- 5488 UGUAAGGUUUGUU 3296-3318 1251428.2 2337677.1 CCUUACA 2337678.1 UCACAUAAUU AD- A- 5473 UAUGUGAAACAAACC 3299-3319 A- 5489 UCGUAAGGUUUGU 3297-3319 1251434.2 2337679.1 UUACGA 2337687.1 UUCACAUAGU

TABLE 16 SCN9A Lipid-Conjugated Modified Sequences. The C16 modifications shown are exemplary modifications. It is understood other lipophilic moieties may be used at other locations within the duplex as provided above. SEQ SEQ Duplex ID ID Name Modified sense strand sequence NO: Modified antisense strand sequence NO: AD-1479539 gscscca(Ahd)AfaUfAfCfugauaauasgsa 5490 VPusCfsuauUfaUfCfaguaUfuUfugggcsasg 5645 AD-1479540 asasggg(Ahd)AfaAfCfAfaucuuccgsusa 5491 VPusAfscggAfaGfAfuuguUfuUfcccuususg 5646 AD-1479541 ususugu(Ahd)gadTcdTugcaauuascsa 5492 VPusdGsuadAudTgcaadGadTcdTacaaasasg 5647 AD-1479542 asusguc(Ghd)AfgUfAfCfacuuuuacsusa 5493 VPusAfsguaAfaAfGfuguaCfuCfgacaususu 5648 AD-1479543 csusaaa(Uhd)UfaUfGfGfaaguaaucsusa 5494 VPusAfsgauUfaCfUfuccaUfaAfuuuagsgsa 5649 AD-1479544 usgsaga(Chd)UfgAfCfAfcauuguaasusa 5495 VPusAfsuuaCfaAfUfguguCfaGfucucasasg 5650 AD-1479545 asuscuu(Chd)uudTgdTcguagugasusa 5496 VPusdAsucdAcdTacgadCadAadGaagauscsa 5651 AD-1479546 usgsguu(Uhd)CfaGfCfAfcagauucasgsa 5497 VPusCfsugaAfuCfUfgugcUfgAfaaccascsa 5652 AD-1479547 ascsaug(Ahd)ucdTudCuuugucgusasa 5498 VPusdTsacdGadCaaagdAadGadTcaugusasg 5653 AD-1479548 csusucu(Ghd)AfaAfCfAfuccaaacusgsa 5499 VPusCfsaguUfuGfGfauguUfuCfagaagsasa 5654 AD-1479549 usasuug(Uhd)GfaCfUfUfuaaguuuasgsa 5500 VPusCfsuaaAfcUfUfaaagUfcAfcaauasasg 5655 AD-1479550 csasccu(Uhd)CfuCfCfUfuaaaauucsusa 5501 VPusAfsgaaUfuUfUfaaggAfgAfaggugsasc 5656 AD-1479551 ususgug(Ahd)CfuUfUfAfaguuuagusgsa 5502 VPusCfsacuAfaAfCfuuaaAfgUfcacaasusa 5657 AD-1479552 gsasucu(Uhd)cudTudGucguagugsasa 5503 VPusdTscadCudAcgacdAadAgdAagaucsasu 5658 AD-1479553 ususgcu(Ahd)UfaGfGfAfaauuugguscsa 5504 VPusGfsaccAfaAfUfuuccUfaUfagcaasgsu 5659 AD-1479554 csasuga(Uhd)CfuUfCfUfuugucguasgsa 5505 VPusCfsuacGfaCfAfaagaAfgAfucaugsusa 5660 AD-1479555 ususgau(Ahd)GfuUfAfCfcuaguuugscsa 5506 VPusGfscaaAfcUfAfgguaAfcUfaucaasasa 5661 AD-1479556 ususcug(Uhd)GfuAfGfGfagaauucascsa 5507 VPusGfsugaa(Tgn)ucuccuAfcAfcagaasgsc 5662 AD-1479557 usgscua(Uhd)agdGadAauuuggucsusa 5508 VPusdAsgadCcdAaauudTcdCudAuagcasasg 5663 AD-1479558 ususcug(Uhd)gudAgdGagaauucascsa 5509 VPusdGsugdAadTucucdCudAcdAcagaasgsc 5664 AD-1479559 usgsaua(Ghd)UfuAfCfCfuaguuugcsasa 5510 VPusUfsgcaAfaCfUfagguAfaCfuaucasasa 5665 AD-1479560 gsusuug(Ahd)AfcAfCfAfaaucuuucsgsa 5511 VPusCfsgaaAfgAfUfuuguGfuUfcaaacscsu 5666 AD-1479561 gsasgau(Ghd)GfaUfUfCfucuucguuscsa 5512 VPusGfsaacGfaAfGfagaaUfcCfaucucscsc 5667 AD-1479562 asusgau(Chd)UfuCfUfUfugucguagsusa 5513 VPusAfscuaCfgAfCfaaagAfaGfaucausgsu 5668 AD-1479563 asgscuu(Ghd)AfaGfUfAfaaauuagascsa 5514 VPusGfsucuAfaUfUfuuacUfuCfaagcususa 5669 AD-1479564 asuscuu(Chd)UfuUfGfUfcguagugasusa 5515 VPusAfsucaCfuAfCfgacaAfaGfaagauscsa 5670 AD-1479565 usgsauc(Uhd)ucdTudTgucguagusgsa 5516 VPusdCsacdTadCgacadAadGadAgaucasusg 5671 AD-1479566 asuscug(Ahd)gadCudGaauuugccsgsa 5517 VPusdCsggdCadAauucdAgdTcdTcagauscsc 5672 AD-1479567 asusgau(Chd)uudCudTugucguagsusa 5518 VPusdAscudAcdGacaadAgdAadGaucausgsu 5673 AD-1479568 csasagu(Ghd)UfuCfCfUfacugucausgsa 5519 VPusCfsaugAfcAfGfuaggAfaCfacuugsasa 5674 AD-1479569 asusgug(Ahd)AfaCfAfAfaccuuacgsusa 5520 VPusAfscguAfaGfGfuuugUfuUfcacausasa 5675 AD-1479570 usgsucg(Ahd)gudAcdAcuuuuacusgsa 5521 VPusdCsagdTadAaagudGudAcdTcgacasusu 5676 AD-1479571 usgsuag(Ghd)AfgAfAfUfucacuuuuscsa 5522 VPusGfsaaaAfgUfGfaauuCfuCfcuacascsa 5677 AD-1479572 gsgscgu(Uhd)GfuAfGfUfuccuaucuscsa 5523 VPusGfsagaUfaGfGfaacuAfcAfacgccsusu 5678 AD-1479573 usasuug(Uhd)gadCudTuaaguuuasgsa 5524 VPusdCsuadAadCuuaadAgdTcdAcaauasasg 5679 AD-1479574 ususgug(Ahd)cudTudAaguuuagusgsa 5525 VPusdCsacdTadAacuudAadAgdTcacaasusa 5680 AD-1209344 csasaca(Chd)aadTudTcuucuuagscsa 5526 VPusdGscudAadGaagadAadTudGuguugsusu 5681 AD-1479575 asasggg(Ahd)aadAcdAaucuuccgsusa 5527 VPusdAscgdGadAgauudGudTudTcccuususg 5682 AD-1331347 usgsucg(Ahd)GfuAfCfAfcuuuuacusgsa 5528 VPusCfsaguAfaAfAfguguAfcUfcgacasusu 5683 AD-1479576 gsasucu(Uhd)CfuUfUfGfucguagugsasa 5529 VPusUfscacUfaCfGfacaaAfgAfagaucsasu 5684 AD-1443073 asgscau(Ahd)AfaUfGfUfuuucgaaasusa 5530 VPusAfsuuuCfgAfAfaacaUfuUfaugcususc 5685 AD-1479577 usasugu(Ghd)AfaAfCfAfaaccuuacsgsa 5531 VPusCfsguaAfgGfUfuuguUfuCfacauasasu 5686 AD-1479578 usgsuag(Ghd)agdAadTucacuuuuscsa 5532 VPusdGsaadAadGugaadTudCudCcuacascsa 5687 AD-1479579 csasuga(Uhd)cudTcdTuugucguasgsa 5533 VPusdCsuadCgdAcaaadGadAgdAucaugsusa 5688 AD-1183928 ususcug(Uhd)GfuAfGfGfagaauucascsa 5534 VPusGfsugaAfuUfCfuccuAfcAfcagaasgsc 5689 AD-1183930 ususugu(Ahd)GfaUfCfUfugcaauuascsa 5535 VPusGfsuaaUfuGfCfaagaUfcUfacaaasasg 5690 AD-1331355 usgsucgaguAfCfAfcuuu(Uhd)acusgsa 5536 VPusCfsagdTadAaaguguAfcUfcgacasusu 5691 AD-1479580 uscsgaguAfCfAfcuuu(Uhd)acusgsa 5537 VPusCfsagdTadAaaguguAfcUfcgascsg 5692 AD-1331354 csasuga(Uhd)cuUfCfUfuugucguasgsa 5538 VPuCfuadCgdAcaaadGadAgdAucaugsusg 5693 AD-1479581 gsasaaa(Chd)aaUfCfUfuccauuucsasa 5539 VPuUfgadAadTggaagaUfudGuuuucscsc 5694 AD-1331351 gsasaaa(Chd)aaUfCfUfuccguuucsasa 5540 VPuUfgadAa(C2p)ggaagaUfudGuuuucscsc 5695 AD-1479582 asasaacaAfuCfUfUfccgu(Uhd)ucasasa 5541 VPusUfsugdAadAcggaagAfuUfguuuuscsc 5696 AD-1331350 asasaacaauCfUfUfccgu(Uhd)ucasasa 5542 VPuUfugdAadAcggadAgdAuUfguuuuscsc 5697 AD-1479583 gsgscuu(Chd)UfgUfgUfaggagaaususa 5543 VPudAaudTc(Tgn)ccuadCaCfadGaagccsusc 5698 AD-1479584 ususcug(Uhd)guAfgdGagaauucascsa 5544 VPusdGsugdAa(U2p)ucucdCuAfcAfcagaasg 5699 sc AD-1479585 gsusguaggadGadAuuca(Chd)uuususa 5545 VPudAaadAgdTgaaudTcUfcCfuacacsgsg 5700 AD-1479586 usgsuaggagdAaUfucau(Uhd)uuuscsa 5546 VPusdGsaadAadAugaadTuCfuCfcuacascsg 5701 AD-1479587 gsgsagaaUfuCfAfCfuuuu(Chd)uucsgsa 5547 VPusCfsgadAgdAaaagugAfaUfucuccsusg 5702 AD-1479588 gsgsagaaUfuCfaCfuuuu(Chd)uucsgsa 5548 VPuCfgadAgdAaaagdTgdAaUfucuccsusg 5703 AD-1479589 csusgaagCfaUfAfAfaugu(Uhd)uucsgsa 5549 VPusCfsgadAadAcauuuaUfgCfuucagsgsu 5704 AD-1479590 usgsaag(Chd)audAadAuguuuucgsasa 5550 VPuUfcgdAadAacaudTuAfudGcuucasgsg 5705 AD-1479591 gsasagcaUfaAfAfUfguuu(Uhd)cgasasa 5551 VPusUfsucdGadAaacauuUfaUfgcuucsasg 5706 AD-1331349 gsasagcauadAaUfguuu(Uhd)cgasasa 5552 VPuUfucdGadAaacadTuUfaUfgcuucsasg 5707 AD-1479592 asasgca(Uhd)aadAudGuuuucgaasasa 5553 VPuUfuudCgdAaaacdAuUfudAugcuuscsg 5708 AD-1479593 asgsca(Uhd)aaaUfgUfuuucgaaasusa 5554 VPudAuudTcdGaaaadCaUfuUfaugcususc 5709 AD-1479594 asgscauaAfaUfGfUfuuu(Chd)gaaasusa 5555 VPusAfsuuuCfgAfAfaacaUfuUfaugcususc 5710 AD-1479595 asgscauaAfaUfGfUfuuu(Chd)gaaasusa 5556 VPusAfsuudTc(G2p)aaaacaUfuUfaugcususc 5711 AD-1479596 asgscauaaaUfgUfuuu(Uhd)gaaasusa 5557 VPusAfsuudTcdAaaaadCaUfuUfaugcususc 5712 AD-1479597 asgscauaaaUfgUfuuu(Chd)gaaasusa 5558 VPusdAsuudTc(G2p)aaaadCaUfuUfaugcusc 5713 sc AD-1479598 csasuaaaUfgUfuuu(Chd)gaaasusa 5559 VPusdAsuudTc(G2p)aaaadCaUfuUfaugscsu 5714 AD-1479599 asgscauaaaUfgUfuuu(Chd)gaaasusa 5560 VPudAuudTc(G2p)aaaadCaUfuUfaugcususc 5715 AD-1479600 gscsa(Uhd)aaaugUfUfuucgaaaususa 5561 VPusdAsaudTu(C2p)gaaaacAfuUfuaugcsusu 5716 AD-1479601 asusaaa(Uhd)guUfUfUfcgaaauucsasa 5562 VPusUfsgadAudTucgaaaAfcAfuuuausgsc 5717 AD-1479602 asusaaa(Uhd)guUfUfUfcgaaauucsasa 5563 VPusUfsgadAudTucgaaaAfcAfuuuausgsu 5718 AD-1479603 usasaaugUfuUfuCfgaaa(Uhd)ucascsa 5564 VPudGugdAadTuucgdAadAaCfauuuasusg 5719 AD-1479604 usasca(Uhd)gAfuCfUfUfcuuugucgsusa 5565 VPusAfscgdAcdAaagaagAfuCfauguasgsg 5720 AD-1479605 csasuga(Uhd)CfuUfCfUfuugucguasgsa 5566 VPusCfsuadCgdAcaaagaAfgAfucaugsusg 5721 AD-1479606 csasuga(Uhd)CfuUfCfUfuugucguasgsa 5567 VPuCfuadCgdAcaaadGadAgdAucaugsusg 5722 AD-1331348 asusgau(Chd)UfuCfUfUfugucguagsusa 5568 VPudAcudAcdGacaadAgdAadGaucausgsu 5723 AD-1479607 usgsauc(Uhd)UfcUfUfUfgucguagusgsa 5569 VPudCacdTadCgacadAadGadAgaucasusg 5724 AD-1479608 uscsu(Uhd)CfuUfudGucguagugsasa 5570 VPusUfscadCu(Agn)cgacdAaAfgAfagasusc 5725 AD-1479609 uscsu(Uhd)CfuUfudGucguagugsasa 5571 VPusUfscadCu(A2p)cgacdAaAfgAfagasusc 5726 AD-1443072 gsasucu(Uhd)CfuUfudGucguagugsasa 5572 VPuUfcadCu(A2p)cgacdAaAfgAfagaucsgsu 5727 AD-1479610 gsasucu(Uhd)CfuUfudGUfcguagugsasa 5573 VPuUfcadCu(A2p)cgacdAaAfgAfagaucsgsu 5728 AD-1479611 gsasucu(Uhd)CfuUfUfgUfCfguagugsasa 5574 VPuUfcadCu(A2p)cgacaaAfgAfagaucsgsu 5729 AD-1479612 uscsuuugUfcgUfAfguga(Uhd)uuuscsa 5575 VPudGaadAadTcacudAcdGaCfaaagasgsg 5730 AD-1479613 asusccu(Uhd)UfugUfAfgaucuugcsasa 5576 VPusUfsgcdAa(G2p)aucuacAfaAfaggauscsc 5731 AD-1479614 cscsuuu(Uhd)gudAgdAucuugcaasusa 5577 VPudAuudGc(A2p)agaudCuAfcAfaaaggsgsu 5732 AD-1479615 csusuuugUfagAfUfcuug(Chd)aaususa 5578 VPusAfsaudTg(C2p)aagaucUfaCfaaaagsgsg 5733 AD-1479616 ususuug(Uhd)AfgAfUfCfuugcaauusasa 5579 VPusUfsaadTu(G2p)caagauCfuAfcaaaasgsg 5734 AD-1479617 ususuug(Uhd)agAfUfCfuugcaauusasa 5580 VPusUfsaadTu(G2p)caagauCfuAfcaaagscsc 5735 AD-1479618 ususug(Uhd)agaUfCfUfugcaauuascsa 5581 VPusGfsuaaUfuGfCfaagaUfcUfacaaasgsg 5736 AD-1479619 ususug(Uhd)agaUfCfUfugcaauuascsa 5582 VPusdGsuadAu(Tgn)gcaagaUfcUfacaaasgsg 5737 AD-1479620 ususug(Uhd)agaUfCfUfugcaauuascsa 5583 VPudGuadAu(Tgn)gcaagaUfcUfacaaasgsg 5738 AD-1479621 ususguagauCfUfUfgcaa(Uhd)uacscsa 5584 VPusdGsgudAa(U2p)ugcaagAfuCfuacaasgsg 5739 AD-1479622 gsusaga(Uhd)CfuUfgCfaauuaccasusa 5585 VPudAugdGudAauugdCaAfgAfucuacsgsg 5740 AD-1479623 asasuua(Uhd)gudGadAacaaaccususa 5586 VPudAagdGu(U2p)uguudTcAfcAfuaauususg 5741 AD-1479624 asusuaugugdAadAcaaa(Chd)cuusasa 5587 VPuUfaadGg(Tgn)uugudTuCfaCfauaaususu 5742 AD-1479625 ususaug(Uhd)gaAfAfCfaaaccuuascsa 5588 VPudGuadAg(G2p)uuuguuUfcAfcauaasusu 5743 AD-1331354 csasuga(Uhd)cuUfCfUfuugucguasgsa 5589 VPuCfuadCgdAcaaadGadAgdAucaugsusg 5744 AD-1479581 gsasaaa(Chd)aaUfCfUfuccauuucsasa 5590 VPuUfgadAadTggaagaUfudGuuuucscsc 5745 AD-1331351 gsasaaa(Chd)aaUfCfUfuccguuucsasa 5591 VPuUfgadAa(C2p)ggaagaUfudGuuuucscsc 5746 AD-1331350 asasaacaauCfUfUfccgu(Uhd)ucasasa 5592 VPuUfugdAadAcggadAgdAuUfguuuuscsc 5747 AD-1479583 gsgscuu(Chd)UfgUfgUfaggagaaususa 5593 VPudAaudTc(Tgn)ccuadCaCfadGaagccsusc 5748 AD-1479585 gsusguaggadGadAuuca(Chd)uuususa 5594 VPudAaadAgdTgaaudTcUfcCfuacacsgsg 5749 AD-1479588 gsgsagaaUfuCfaCfuuuu(Chd)uucsgsa 5595 VPuCfgadAgdAaaagdTgdAaUfucuccsusg 5750 AD-1479590 usgsaag(Chd)audAadAuguuuucgsasa 5596 VPuUfcgdAadAacaudTuAfudGcuucasgsg 5751 AD-1331349 gsasagcauadAaUfguuu(Uhd)cgasasa 5597 VPuUfucdGadAaacadTuUfaUfgcuucsasg 5752 AD-1479592 asasgca(Uhd)aadAudGuuuucgaasasa 5598 VPuUfuudCgdAaaacdAuUfudAugcuuscsg 5753 AD-1479593 asgsca(Uhd)aaaUfgUfuuucgaaasusa 5599 VPudAuudTcdGaaaadCaUfuUfaugcususc 5754 AD-1479599 asgscauaaaUfgUfuuu(Chd)gaaasusa 5600 VPudAuudTc(G2p)aaaadCaUfuUfaugcususc 5755 AD-1479603 usasaaugUfuUfuCfgaaa(Uhd)ucascsa 5601 VPudGugdAadTuucgdAadAaCfauuuasusg 5756 AD-1479606 csasuga(Uhd)CfuUfCfUfuugucguasgsa 5602 VPuCfuadCgdAcaaadGadAgdAucaugsusg 5757 AD-1331348 asusgau(Chd)UfuCfUfUfugucguagsusa 5603 VPudAcudAcdGacaadAgdAadGaucausgsu 5758 AD-1479607 usgsauc(Uhd)UfcUfUfUfgucguagusgsa 5604 VPudCacdTadCgacadAadGadAgaucasusg 5759 AD-1443072 gsasucu(Uhd)CfuUfudGucguagugsasa 5605 VPuUfcadCu(A2p)cgacdAaAfgAfagaucsgsu 5760 AD-1479610 gsasucu(Uhd)CfuUfudGUfcguagugsasa 5606 VPuUfcadCu(A2p)cgacdAaAfgAfagaucsgsu 5761 AD-1479611 gsasucu(Uhd)CfuUfUfgUfCfguagugsasa 5607 VPuUfcadCu(A2p)cgacaaAfgAfagaucsgsu 5762 AD-1479612 uscsuuugUfcgUfAfguga(Uhd)uuuscsa 5608 VPudGaadAadTcacudAcdGaCfaaagasgsg 5763 AD-1479614 cscsuuu(Uhd)gudAgdAucuugcaasusa 5609 VPudAuudGc(A2p)agaudCuAfcAfaaaggsgsu 5764 AD-1479620 ususug(Uhd)agaUfCfUfugcaauuascsa 5610 VPudGuadAu(Tgn)gcaagaUfcUfacaaasgsg 5765 AD-1479622 gsusaga(Uhd)CfuUfgCfaauuaccasusa 5611 VPudAugdGudAauugdCaAfgAfucuacsgsg 5766 AD-1479623 asasuua(Uhd)gudGadAacaaaccususa 5612 VPudAagdGu(U2p)uguudTcAfcAfuaauususg 5767 AD-1479624 asusuaugugdAadAcaaa(Chd)cuusasa 5613 VPuUfaadGg(Tgn)uugudTuCfaCfauaaususu 5768 AD-1479625 ususaug(Uhd)gaAfAfCfaaaccuuascsa 5614 VPudGuadAg(G2p)uuuguuUfcAfcauaasusu 5769 AD-1481938 gsusaga(Uhd)CfuUfgCfaauuaccasusa 5615 VPusdAsugdGudAauugdCaAfgAfucuacsgsg 5770 AD-1481939 asasuua(Uhd)gudGadAacaaaccususa 5616 VPusdAsagdGu(U2p)uguudTcAfcAfuaauusu 5771 sg AD-1481940 asusuaugugdAadAcaaa(Chd)cuusasa 5617 VPusUfsaadGg(Tgn)uugudTuCfaCfauaausu 5772 su AD-1481941 ususaug(Uhd)gaAfAfCfaaaccuuascsa 5618 VPusdGsuadAg(G2p)uuuguuUfcAfcauaasusu 5773 AD-1481942 csasuga(Uhd)cuUfCfUfuugucguasgsa 5619 VPusCfsuadCgdAcaaadGadAgdAucaugsusg 5774 AD-1481943 gsasaaa(Chd)aaUfCfUfuccauuucsasa 5620 VPusUfsgadAadTggaagaUfudGuuuucscsc 5775 AD-1481944 gsasaaa(Chd)aaUfCfUfuccguuucsasa 5621 VPusUfsgadAa(C2p)ggaagaUfudGuuuucscsc 5776 AD-1481945 asasaacaauCfUfUfccgu(Uhd)ucasasa 5622 VPusUfsugdAadAcggadAgdAuUfguuuuscsc 5777 AD-1481946 gsgscuu(Chd)UfgUfgUfaggagaaususa 5623 VPusdAsaudTc(Tgn)ccuadCaCfadGaagccsu 5778 sc AD-1481947 gsusguaggadGadAuuca(Chd)uuususa 5624 VPusdAsaadAgdTgaaudTcUfcCfuacacsgsg 5779 AD-1481948 gsgsagaaUfuCfaCfuuuu(Chd)uucsgsa 5625 VPusCfsgadAgdAaaagdTgdAaUfucuccsusg 5780 AD-1481949 usgsaag(Chd)audAadAuguuuucgsasa 5626 VPuslIfscgdAadAacaudTuAfudGcuucasgsg 5781 AD-1481950 gsasagcauadAaUfguuu(Uhd)cgasasa 5627 VPusUfsucdGadAaacadTuUfaUfgcuucsasg 5782 AD-1481951 asasgca(Uhd)aadAudGuuuucgaasasa 5628 VPusUfsuudCgdAaaacdAuUfudAugcuuscsg 5783 AD-1481952 asgsca(Uhd)aaaUfgUfuuucgaaasusa 5629 VPusdAsuudTcdGaaaadCaUfuUfaugcususc 5784 AD-1481953 asgscauaaaUfgUfuuu(Chd)gaaasusa 5630 VPusdAsuudTc(G2p)aaaadCaUfuUfaugcusu 5785 sc AD-1481954 usasaaugUfuUfuCfgaaa(Uhd)ucascsa 5631 VPusdGsugdAadTuucgdAadAaCfauuuasusg 5786 AD-1481955 csasuga(Uhd)CfuUfCfUfuugucguasgsa 5632 VPusCfsuadCgdAcaaadGadAgdAucaugsusg 5787 AD-1481956 asusgau(Chd)UfuCfUfUfugucguagsusa 5633 VPusdAscudAcdGacaadAgdAadGaucausgsu 5788 AD-1481957 usgsauc(Uhd)UfcUfUfUfgucguagusgsa 5634 VPusdCsacdTadCgacadAadGadAgaucasusg 5789 AD-1481958 gsasucu(Uhd)CfuUfudGucguagugsasa 5635 VPusUfscadCu(A2p)cgacdAaAfgAfagaucsg 5790 su AD-1481959 gsasucu(Uhd)CfuUfudGUfcguagugsasa 5636 VPusUfscadCu(A2p)cgacdAaAfgAfagaucsg 5791 su AD-1481960 gsasucu(Uhd)CfuUfUfgUfCfguagugsasa 5637 VPusUfscadCu(A2p)cgacaaAfgAfagaucsgsu 5792 AD-1481961 uscsuuugUfcgUfAfguga(Uhd)uuuscsa 5638 VPusdGsaadAadTcacudAcdGaCfaaagasgsg 5793 AD-1481962 cscsuuu(Uhd)gudAgdAucuugcaasusa 5639 VPusdAsuudGc(A2p)agaudCuAfcAfaaaggsg 5794 su AD-1479619 ususug(Uhd)agaUfCfUfugcaauuascsa 5640 VPusdGsuadAu(Tgn)gcaagaUfcUfacaaasgsg 5795 AD-1481938 gsusaga(Uhd)CfuUfgCfaauuaccasusa 5641 VPusdAsugdGudAauugdCaAfgAfucuacsgsg 5796 AD-1481939 asasuua(Uhd)gudGadAacaaaccususa 5642 VPusdAsagdGu(U2p)uguudTcAfcAfuaauusu 5797 sg AD-1481940 asusuaugugdAadAcaaa(Chd)cuusasa 5643 VPusUfsaadGg(Tgn)uugudTuCfaCfauaausu 5798 su AD-1481941 ususaug(Uhd)gaAfAfCfaaaccuuascsa 5644 VPusdGsuadAg(G2p)uuuguuUfcAfcauaasusu 5799 AD-1331352 usgsucgaguAfCfAfcuuu(Uhd)acusgsa 5800 VPusCfsagdTadAaagudGuAfcdTcgacasusu 5801

Example 2. In Vitro Screening of SCN9A siRNA

Experimental Methods

Dual-Glo® Luciferase Assay

Hepa1-6 cells (ATCC) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. A single-dose experiment was performed at 10 nM final duplex concentration. Three different siRNA and psiCHECK2-SCN9A plasmid transfections were carried out with each plasmid containing the 3′ untranslated region (UTR). The three plasmids were referred to as SCN9A-1, SCN9A-2, and SCN9A-3. Transfection was carried out by adding 10 nM of siRNA duplexes and 30-75 ng of one of the three psiCHECK2-SCN9A plasmids per well along with 4.9 μL of Opti-MEM plus 0.5 μL of Lipofectamine 2000 per well (Invitrogen, Carlsbad Calif. cat #13778-150) and then incubated at room temperature for 15 minutes. The mixture was then added to the cells (approximately 15,000 per well), which were re-suspended in 35 μL of fresh complete media. The transfected cells were incubated at 37° C. in an atmosphere of 5% CO₂.

Twenty-four hours after the siRNAs and psiCHECK2-SCN9A plasmid were transfected; Firefly (transfection control) and Renilla (fused to SCN9A target sequence) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 20 μL of Dual-Glo® Luciferase Reagent (Promega) equal to the culture medium volume to each well and mixing. The mixture was incubated at room temperature for 30 minutes before luminescence (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 μL of room temperature of Dual-Glo® Stop & Glo® Reagent (Promega) were added to each well and the plates were incubated for 10-15 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenched the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (SCN9A) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-SCN9A targeting siRNA. All transfections are done with n=4.

Results

The results of the single-dose dual luciferase screen in Hepa1-6 cells transfected with either the SCN9A-1 (added at 30 ng/well), SCN9A-2 (added at 75 ng/well), or SCN9A-3 plasmid (added at 30 ng/well) and treated with an exemplary set of SCN9A siRNAs is shown in Table 3 (correspond to siRNAs in Table 2A). The single-dose experiment was performed at a 10 nM final duplex concentration and the data are expressed as percent SCN9A luciferase signal remaining relative to cells treated with a non-targeting control.

Of the siRNA duplexes evaluated in cells transfected with SCN9A-1, 2 achieved ≥80% knockdown of SCN9A, 34 achieved ≥60% knockdown of SCN9A, 92 achieved ≥30% knockdown of SCN9A, and 95 achieved ≥20% knockdown of SCN9A.

Of the siRNA duplexes evaluated in cells transfected with SCN9A-2, 9 achieved ≥80% knockdown of SCN9A, 90 achieved ≥60% knockdown of SCN9A, 130 achieved ≥30% knockdown of SCN9A, and 132 achieved ≥20% knockdown of SCN9A.

Of the siRNA duplexes evaluated in cells transfected with SCN9A-3, 7 achieved ≥60% knockdown of SCN9A, 34 achieved ≥30% knockdown of SCN9A, and 47 achieved ≥20% knockdown of SCN9A.

TABLE 3 SCN9A in vitro dual luciferase 10 nM screen with one set of exemplary human SCN9A siRNAs 10 nM % of SCN9A Luciferase Duplex ID* Plasmid signal Remaining StDev AD-887232.1 SCN9A-1 93.8 0.080 AD-887233.1 20.6 0.038 AD-887234.1 42.8 0.086 AD-887235.1 20.6 0.035 AD-887236.1 21.9 0.037 AD-887237.1 24.8 0.018 AD-887238.1 57.6 0.040 AD-887239.1 28.7 0.014 AD-887240.1 60.8 0.043 AD-887241.1 35.2 0.014 AD-887242.1 58.7 0.092 AD-887243.1 65.6 0.099 AD-887244.1 23.7 0.019 AD-887245.1 15.9 0.020 AD-887246.1 20.4 0.022 AD-887247.1 20.1 0.018 AD-887248.1 19.9 0.011 AD-887249.1 24.1 0.045 AD-887250.1 31.5 0.039 AD-887251.1 27.1 0.040 AD-887252.1 22.4 0.026 AD-887253.1 23.1 0.015 AD-887254.1 24.6 0.033 AD-887255.1 44.5 0.072 AD-887256.1 51.4 0.082 AD-887257.1 21.8 0.025 AD-887258.1 51.7 0.124 AD-887259.1 30.2 0.046 AD-887260.1 26.8 0.043 AD-887261.1 27.9 0.030 AD-887262.1 33.3 0.094 AD-887263.1 40.6 0.042 AD-887264.1 31.2 0.047 AD-887265.1 37.0 0.045 AD-887266.1 44.5 0.131 AD-887267.1 46.4 0.059 AD-887268.1 36.7 0.035 AD-887269.1 35.2 0.038 AD-887270.1 34.1 0.046 AD-887271.1 71.6 0.036 AD-887272.1 49.4 0.018 AD-887273.1 50.6 0.041 AD-887274.1 36.7 0.099 AD-887275.1 89.3 0.041 AD-887276.1 49.8 0.036 AD-887277.1 97.5 0.152 AD-887278.1 49.3 0.052 AD-887279.1 86.3 0.086 AD-887280.1 45.5 0.025 AD-887281.1 42.7 0.086 AD-887282.1 105.3 0.240 AD-887283.1 121.4 0.208 AD-887284.1 82.6 0.116 AD-887285.1 54.7 0.147 AD-887286.1 122.0 0.057 AD-887287.1 44.2 0.090 AD-887288.1 40.7 0.026 AD-887289.1 54.0 0.083 AD-887290.1 51.4 0.094 AD-887291.1 52.5 0.112 AD-887292.1 37.5 0.061 AD-887293.1 41.8 0.083 AD-887294.1 103.6 0.109 AD-887295.1 46.0 0.100 AD-887296.1 60.7 0.049 AD-887297.1 42.3 0.072 AD-887298.1 47.6 0.035 AD-887299.1 65.9 0.068 AD-887300.1 89.9 0.040 AD-887301.1 66.6 0.078 AD-887302.1 59.6 0.053 AD-887303.1 31.3 0.032 AD-887304.1 37.5 0.055 AD-887305.1 73.2 0.056 AD-887306.1 35.5 0.021 AD-887307.1 36.7 0.032 AD-887308.1 97.6 0.098 AD-887309.1 60.5 0.066 AD-887310.1 45.8 0.018 AD-887311.1 40.8 0.037 AD-887312.1 44.9 0.113 AD-887313.1 48.3 0.077 AD-887314.1 45.3 0.056 AD-887315.1 44.2 0.029 AD-887316.1 55.0 0.054 AD-887317.1 51.3 0.045 AD-887318.1 55.8 0.053 AD-887319.1 44.2 0.020 AD-887320.1 50.9 0.060 AD-887321.1 50.3 0.093 AD-887322.1 104.1 0.129 AD-887323.1 99.3 0.064 AD-887324.1 94.8 0.083 AD-887325.1 36.2 0.063 AD-887326.1 42.4 0.033 AD-887327.1 57.2 0.104 AD-887328.1 57.9 0.036 AD-887329.1 65.0 0.124 AD-887330.1 61.0 0.026 AD-887331.1 89.2 0.079 AD-887332.1 44.3 0.078 AD-887333.1 42.7 0.135 AD-887334.1 57.7 0.035 AD-887335.1 59.8 0.088 AD-887336.1 75.3 0.098 AD-887337.1 47.5 0.080 AD-887338.1 51.8 0.056 AD-887339.1 60.2 0.068 AD-887340.1 126.3 0.223 AD-887341.1 109.1 0.127 AD-887342.1 53.1 0.101 AD-887343.1 55.3 0.042 AD-887344.1 SCN9A-2 13.5 0.039 AD-887345.1 21.4 0.006 AD-887346.1 13.0 0.026 AD-887347.1 27.0 0.041 AD-887348.1 34.9 0.039 AD-887349.1 13.6 0.045 AD-887350.1 18.4 0.033 AD-887351.1 12.8 0.021 AD-887352.1 14.6 0.034 AD-887353.1 84.0 0.081 AD-887354.1 12.8 0.028 AD-887355.1 25.5 0.051 AD-887356.1 17.7 0.040 AD-887357.1 17.3 0.009 AD-887358.1 22.6 0.033 AD-887359.1 35.0 0.032 AD-887360.1 23.8 0.048 AD-887361.1 21.7 0.026 AD-887362.1 21.5 0.027 AD-887363.1 25.6 0.044 AD-887364.1 33.5 0.038 AD-887365.1 28.2 0.037 AD-887366.1 25.6 0.015 AD-887367.1 23.4 0.028 AD-887368.1 21.5 0.033 AD-887369.1 30.8 0.032 AD-887370.1 28.8 0.034 AD-887371.1 27.6 0.050 AD-887372.1 27.0 0.053 AD-887373.1 39.0 0.042 AD-887374.1 78.8 0.037 AD-887375.1 37.0 0.056 AD-887376.1 30.0 0.069 AD-887377.1 28.1 0.032 AD-887378.1 20.8 0.025 AD-887379.1 26.2 0.023 AD-887380.1 39.9 0.086 AD-887381.1 34.5 0.007 AD-887382.1 25.5 0.027 AD-887383.1 29.2 0.040 AD-887384.1 27.2 0.043 AD-887385.1 33.6 0.044 AD-887386.1 29.4 0.020 AD-887387.1 28.8 0.060 AD-887388.1 45.3 0.087 AD-887389.1 32.6 0.062 AD-887390.1 27.0 0.055 AD-887391.1 43.3 0.039 AD-887392.1 35.7 0.019 AD-887393.1 30.5 0.017 AD-887394.1 33.3 0.022 AD-887395.1 32.9 0.051 AD-887396.1 39.5 0.028 AD-887397.1 33.7 0.040 AD-887398.1 37.0 0.020 AD-887399.1 36.5 0.069 AD-887400.1 42.4 0.042 AD-887401.1 44.3 0.074 AD-887402.1 35.2 0.070 AD-887403.1 35.7 0.027 AD-887404.1 45.5 0.126 AD-887405.1 37.8 0.065 AD-887406.1 36.6 0.064 AD-887407.1 37.9 0.036 AD-887408.1 41.0 0.049 AD-887409.1 39.5 0.044 AD-887410.1 47.0 0.031 AD-887411.1 39.3 0.014 AD-887412.1 34.6 0.052 AD-887413.1 42.1 0.057 AD-887414.1 34.1 0.051 AD-887415.1 32.0 0.036 AD-887416.1 34.1 0.032 AD-887417.1 35.4 0.041 AD-887418.1 42.5 0.078 AD-887419.1 46.2 0.067 AD-887420.1 53.0 0.047 AD-887421.1 37.1 0.025 AD-887422.1 38.0 0.099 AD-887423.1 29.6 0.038 AD-887424.1 44.5 0.077 AD-887425.1 50.5 0.065 AD-887426.1 49.4 0.026 AD-887427.1 38.5 0.067 AD-887428.1 34.0 0.033 AD-887429.1 33.5 0.035 AD-887430.1 33.4 0.046 AD-887431.1 25.2 0.045 AD-887432.1 43.8 0.055 AD-887433.1 34.8 0.043 AD-887434.1 67.0 0.075 AD-887435.1 49.0 0.021 AD-887436.1 35.6 0.099 AD-887437.1 36.8 0.076 AD-887438.1 34.1 0.096 AD-887439.1 32.6 0.031 AD-887440.1 37.9 0.016 AD-887441.1 35.9 0.065 AD-887442.1 46.6 0.085 AD-887443.1 40.5 0.027 AD-887444.1 42.6 0.028 AD-887445.1 63.9 0.129 AD-887446.1 41.0 0.105 AD-887447.1 56.1 0.053 AD-887448.1 37.8 0.101 AD-887449.1 38.8 0.041 AD-887450.1 45.4 0.057 AD-887451.1 61.1 0.024 AD-887452.1 36.3 0.034 AD-887453.1 40.6 0.028 AD-887454.1 45.3 0.081 AD-887455.1 42.7 0.097 AD-887456.1 36.1 0.068 AD-887457.1 54.0 0.057 AD-887458.1 45.0 0.056 AD-887459.1 55.9 0.041 AD-887460.1 37.2 0.023 AD-887461.1 70.7 0.120 AD-887462.1 63.4 0.050 AD-887463.1 28.7 0.015 AD-887464.1 39.9 0.043 AD-887465.1 30.2 0.046 AD-887466.1 43.0 0.056 AD-887467.1 27.8 0.032 AD-887468.1 27.2 0.021 AD-887469.1 49.1 0.052 AD-887470.1 39.3 0.067 AD-887471.1 46.1 0.074 AD-887472.1 40.3 0.071 AD-887473.1 52.3 0.055 AD-887474.1 61.7 0.079 AD-887475.1 55.7 0.020 AD-887476.1 57.8 0.026 AD-887477.1 SCN9A-3 45.3 0.027 AD-887478.1 26.4 0.033 AD-887479.1 69.3 0.083 AD-887480.1 24.3 0.035 AD-887481.1 28.9 0.054 AD-887482.1 32.8 0.077 AD-887483.1 31.6 0.044 AD-887484.1 39.4 0.012 AD-887485.1 38.0 0.044 AD-887486.1 46.3 0.049 AD-887487.1 50.4 0.087 AD-887488.1 47.2 0.076 AD-887489.1 54.9 0.050 AD-887490.1 65.3 0.052 AD-887491.1 74.5 0.080 AD-887492.1 63.8 0.105 AD-887493.1 89.2 0.223 AD-887494.1 43.8 0.085 AD-887495.1 71.7 0.140 AD-887496.1 85.9 0.069 AD-887497.1 72.9 0.025 AD-887498.1 52.9 0.083 AD-887499.1 78.7 0.071 AD-887500.1 70.3 0.062 AD-887501.1 60.9 0.073 AD-887502.1 59.3 0.077 AD-887503.1 55.1 0.068 AD-887504.1 63.9 0.087 AD-887505.1 63.0 0.031 AD-887506.1 62.3 0.062 AD-887507.1 70.9 0.095 AD-887508.1 56.3 0.072 AD-887509.1 78.4 0.065 AD-887510.1 50.9 0.038 AD-887511.1 75.0 0.029 AD-887512.1 81.5 0.154 AD-887513.1 65.7 0.039 AD-887514.1 53.4 0.036 AD-887515.1 55.2 0.084 AD-887516.1 69.7 0.099 AD-887517.1 68.3 0.057 AD-887518.1 91.5 0.134 AD-887519.1 101.2 0.188 AD-887520.1 72.9 0.082 AD-887521.1 75.7 0.048 AD-887522.1 70.8 0.082 AD-887523.1 89.9 0.104 AD-887524.1 54.1 0.062 AD-887525.1 61.5 0.034 AD-887526.1 58.1 0.098 AD-887527.1 66.2 0.138 AD-887528.1 72.5 0.096 AD-887529.1 80.7 0.122 AD-887530.1 73.8 0.018 AD-887531.1 81.8 0.090 (*the number following the decimal point in a duplex name merely refers to a batch production number)

Example 3. In Vitro Screening of SCN9A siRNA

Experimental Methods

Cell Culture and Transfections:

Human neuroblastoma BE(2)-C cells expressing a SC9NA gene were transfected independently by adding 5 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5.1 μl of siRNA duplexes per well into a 384-well plate and are incubated at room temperature for 15 minutes. 40 μl of InVitroGRO CP Medium (BioIVT Cat #Z99029) containing 5×10³ BE(2)-C cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Experiments were performed at 0.1 nM, 1 nM, 10 nM, and 50 nM final duplex concentrations and the results are shown in Table 8.

In a second experiment, BE(2)-C cells expressing a SC9NA gene were transfected independently by adding 5 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5.1 μl of siRNA duplexes per well into a 384-well plate and are incubated at room temperature for 15 minutes. 40 μl of InVitroGRO CP Medium (BioIVT Cat #Z99029) containing 5×10³ BE(2)-C cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Experiments were performed at 0.1 nM, 1 nM, 10 nM, and 50 nM final duplex concentrations and the results are shown in Table 17.

RNA Isolation:

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, Cat #61012). Briefly, 70 μl of Lysis/Binding Buffer and 10 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150 μl Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured and supernatant removed.

cDNA Synthesis:

cDNA was synthesized using ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, Calif., Cat #4368813). 10 μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H2O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 h 37° C.

Real Time PCR:

Two μl of cDNA and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) were added to either 0.5 μl of Human GAPDH TaqMan Probe (4326317E) and 0.5 μl SCN9A Human probe per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). Each duplex was tested at least two times and data were normalized to cells transfected with a non-targeting control siRNA. To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with a non-targeting control siRNA.

Results:

The results of the multi-dose screen in BE(2)-C cells transfected with SCN9A and treated with an exemplary set of SCN9A siRNAs is shown in Table 8 (correspond to siRNAs in Table 4A, 4B, 5A, 5B, 6A, and 6B). The experiment was performed at a 0.1 nM, 1 nM, 10 nM, and 50 nM final duplex concentrations and the data are expressed as percent message remaining relative to non-targeting control.

Of the siRNA duplexes evaluated at 50 nM, 5 achieved ≥80% knockdown of SCN9A, 86 achieved ≥60% knockdown of SCN9A, 266 achieved ≥30% knockdown of SCN9A, 298 achieved ≥20% knockdown of SCN9A, and 314 achieved ≥10% knockdown of SCN9A.

Of the siRNA duplexes evaluated at 10 nM, 2 achieved ≥80% knockdown of SCN9A, 104 achieved ≥60% knockdown of SCN9A, 290 achieved ≥30% knockdown of SCN9A, 316 achieved ≥20% knockdown of SCN9A, and 324 achieved ≥10% knockdown of SCN9A.

Of the siRNA duplexes evaluated at 1 nM, 32 achieved ≥60% knockdown of SCN9A, 203 achieved ≥30% knockdown of SCN9A, 256 achieved ≥20% knockdown of SCN9A, and 296 achieved ≥10% knockdown of SCN9A.

Of the siRNA duplexes evaluated at 0.1 nM, 6 achieved ≥60% knockdown of SCN9A, 111 achieved ≥30% knockdown of SCN9A, 167 achieved ≥20% knockdown of SCN9A, and 213 achieved ≥10% knockdown of SCN9A.

TABLE 8 SCN9A in vitro multidose-dose screen with one set of exemplary human SCN9A siRNA duplexes (*the number following the decimal point in a duplex name merely refers to a batch production number) 50 nM 10 nM 1 nM 0.1 nM % message St. % message St. % message St. % message St. Duplex Name* remaining Dev. remaining Dev. remaining Dev. remaining Dev. AD-1010663.1 20.4 3.1 22.0 3.4 32.2 3.3 29.7 6.2 AD-802123.1 25.2 4.1 31.1 2.6 27.8 2.8 34.8 5.0 AD-961342.1 44.6 9.2 28.8 3.5 46.6 3.0 37.3 1.3 AD-961334.1 40.1 15.1 29.6 3.2 42.0 1.2 39.5 8.9 AD-961179.1 26.1 1.9 28.8 3.1 38.8 1.4 39.6 2.9 AD-1010661.1 22.6 2.8 24.1 5.0 39.5 4.4 39.8 5.5 AD-1010662.1 27.0 4.9 21.1 3.0 44.6 7.0 40.5 7.3 AD-961192.1 29.4 2.2 28.9 2.4 53.5 2.4 40.8 4.0 AD-961189.1 23.7 6.1 24.3 5.6 37.6 1.4 40.9 7.4 AD-961188.1 19.0 0.6 22.0 4.6 34.3 5.6 41.6 1.0 AD-1010665.1 48.8 5.4 36.9 1.7 69.6 4.1 42.2 3.8 AD-802853.2 35.3 3.2 36.9 6.4 40.1 4.2 44.1 5.2 AD-802471.2 23.7 5.7 26.1 4.9 24.5 2.6 44.5 4.4 AD-1010664.1 31.0 3.8 29.9 2.3 54.2 4.4 45.0 5.2 AD-802552.1 27.9 5.0 34.3 2.6 37.9 5.3 45.1 5.5 AD-802625.2 37.8 5.1 36.4 8.7 36.9 4.0 45.2 1.4 AD-802503.1 26.6 1.1 33.7 4.0 34.4 4.0 45.3 6.4 AD-1010700.1 63.7 5.0 32.4 1.4 50.5 1.4 45.4 6.4 AD-961207.1 19.3 1.9 21.0 4.8 43.9 10.2 45.5 10.5 AD-1010671.1 23.7 4.0 26.8 4.0 52.8 7.8 45.6 2.5 AD-1002101.1 33.4 7.3 39.2 3.0 50.1 6.3 45.8 4.9 AD-961208.1 18.6 1.9 20.9 1.5 45.8 10.4 46.6 6.8 AD-1010693.1 44.0 9.9 29.5 2.5 54.2 6.6 46.6 9.2 AD-802553.1 26.7 2.6 32.2 3.2 39.1 5.6 46.9 1.3 AD-961190.1 24.7 1.0 28.4 5.3 49.4 5.0 47.5 4.4 AD-802946.1 29.8 3.8 35.8 4.1 41.1 6.2 48.0 4.5 AD-961191.1 28.3 1.1 29.0 3.0 62.2 5.6 48.9 4.8 AD-801647.1 31.3 4.1 31.6 3.6 45.0 6.6 48.9 6.2 AD-961279.1 42.0 9.7 33.9 5.3 44.2 4.5 50.0 12.3 AD-1010697.1 33.1 4.3 29.8 1.2 54.9 6.5 50.0 15.7 AD-799938.1 26.4 3.5 27.5 8.3 38.6 4.0 51.0 9.2 AD-797636.2 34.7 6.4 25.7 2.7 53.2 9.9 52.4 8.4 AD-961326.1 57.6 9.4 45.9 12.6 48.8 3.2 52.6 7.4 AD-802945.2 46.1 6.1 50.0 3.4 46.1 8.6 52.7 5.3 AD-802206.2 37.4 2.7 43.6 2.8 40.6 9.1 53.0 2.6 AD-1002409.1 40.8 11.4 43.9 2.2 48.4 7.1 53.3 6.3 AD-801263.1 29.9 2.8 33.1 7.1 36.9 4.3 53.4 5.0 AD-961201.1 35.3 3.4 40.8 9.3 66.4 8.6 53.9 7.0 AD-795371.1 22.4 3.7 21.4 1.9 31.9 1.9 54.0 3.8 AD-799587.1 45.6 4.9 40.8 2.1 45.6 4.1 54.4 2.5 AD-802014.1 43.0 2.7 41.3 1.7 46.1 4.2 54.8 4.1 AD-961182.1 32.5 3.1 33.2 5.5 39.8 3.8 54.8 26.8 AD-800966.1 33.8 3.3 36.4 2.5 52.5 2.7 54.9 5.2 AD-795305.1 24.5 1.5 24.8 2.0 38.9 6.0 55.0 9.3 AD-798584.2 45.7 5.5 33.7 3.7 55.1 5.8 55.2 7.5 AD-795366.1 17.1 2.2 19.4 2.7 28.5 3.2 56.2 4.1 AD-1002051.1 38.2 6.7 33.0 5.6 40.3 7.5 56.3 6.6 AD-961321.1 65.9 6.7 33.8 7.4 57.5 5.1 56.7 7.8 AD-797565.2 23.0 1.2 20.6 4.3 43.1 4.8 56.8 3.7 AD-1010698.1 57.0 8.8 30.9 2.7 49.6 2.3 56.8 12.7 AD-799223.1 31.0 3.2 26.8 7.1 36.6 5.3 57.2 5.8 AD-801883.2 42.9 6.3 43.7 8.6 36.9 7.0 57.2 7.5 AD-961155.1 58.5 4.5 50.1 5.2 54.9 9.4 57.5 8.8 AD-1002100.1 45.6 11.2 48.9 6.3 58.7 6.6 57.9 11.8 AD-801658.2 52.9 11.9 51.7 5.9 54.5 5.6 57.9 5.4 AD-800110.1 44.3 8.0 38.6 5.0 44.2 2.1 58.2 3.5 AD-800819.1 32.8 1.7 32.9 6.5 48.0 7.8 58.5 4.3 AD-796618.1 20.8 2.1 23.4 2.2 31.7 3.0 58.6 7.9 AD-797564.2 21.6 3.1 22.7 5.7 48.6 9.8 58.9 7.8 AD-796825.1 13.9 1.4 18.5 2.1 26.3 2.6 59.0 6.8 AD-800297.2 63.1 6.1 51.5 6.4 79.9 8.4 59.3 6.8 AD-801304.1 28.5 2.6 35.0 3.7 46.5 3.5 59.5 3.2 AD-801708.2 48.9 5.3 46.0 7.2 57.8 5.5 59.7 9.0 AD-1010673.1 27.2 4.7 28.8 4.3 52.6 17.0 59.9 8.6 AD-1010699.1 72.3 3.2 33.3 4.7 53.4 2.3 59.9 10.1 AD-1001246.1 40.3 4.9 40.0 1.2 51.9 7.1 60.1 10.8 AD-796209.1 31.6 5.6 26.7 3.0 42.6 3.6 60.5 8.6 AD-801835.1 45.7 2.8 48.9 3.6 55.6 3.5 60.6 3.2 AD-1010677.1 40.4 4.5 49.1 5.8 61.3 13.3 61.1 7.1 AD-802141.2 51.4 7.1 50.8 3.7 54.8 3.8 61.9 1.8 AD-802153.2 67.9 2.2 73.0 4.5 64.5 9.1 63.0 4.7 AD-1010670.1 34.8 4.1 45.0 6.9 58.9 3.2 63.3 15.2 AD-800661.1 42.7 5.7 40.6 2.9 48.7 2.1 63.3 4.1 AD-800058.1 50.9 6.3 42.3 2.5 46.1 1.2 63.8 3.0 AD-795910.1 28.0 1.8 34.2 7.6 39.7 2.7 64.0 5.4 AD-961200.1 52.8 6.6 92.3 36.4 75.1 9.5 64.1 11.1 AD-799939.1 34.8 3.9 36.8 0.3 39.9 3.7 64.1 3.8 AD-1010660.1 42.2 1.5 48.5 9.5 70.2 10.6 64.3 10.0 AD-961093.1 54.9 9.5 53.1 1.9 59.8 4.7 64.4 3.5 AD-1000916.1 42.0 3.3 44.1 2.9 52.6 2.9 64.4 7.3 AD-801681.2 45.4 2.6 43.0 4.6 68.1 5.1 64.5 2.9 AD-995116.1 27.9 6.6 25.8 2.9 48.1 13.4 64.7 3.7 AD-800461.1 40.2 4.1 42.2 1.6 51.0 5.8 64.7 5.6 AD-996318.1 34.1 4.7 23.8 2.0 48.5 7.4 64.8 15.1 AD-795634.2 23.4 6.1 23.2 7.2 46.2 15.6 65.0 9.2 AD-795911.1 26.3 3.0 27.9 4.3 38.4 2.1 65.0 7.0 AD-797036.1 28.3 4.7 27.8 3.2 36.2 3.2 65.1 8.0 AD-961137.1 44.6 5.1 45.9 1.2 63.0 7.1 65.1 5.8 AD-801884.2 41.9 2.3 49.3 3.0 60.9 11.5 65.2 11.4 AD-801490.2 54.0 5.7 56.2 5.9 56.4 6.6 65.3 4.7 AD-802145.2 47.7 1.9 54.3 4.7 45.5 8.3 65.6 10.6 AD-961146.1 54.6 6.1 55.3 2.4 72.6 10.4 65.8 7.3 AD-795909.1 31.2 7.9 30.8 2.9 35.2 2.7 65.9 1.8 AD-1010690.1 75.9 12.7 49.3 12.4 57.5 3.7 66.0 12.8 AD-802071.2 64.9 7.0 58.4 6.1 70.0 2.7 66.2 5.9 AD-795913.1 23.9 3.0 23.3 2.3 35.0 2.5 66.4 8.4 AD-800334.1 37.2 4.7 35.3 6.1 50.8 8.2 66.4 6.1 AD-795739.1 29.0 5.7 24.7 2.4 42.3 6.4 66.6 2.8 AD-796619.1 33.1 2.9 31.4 5.2 39.4 3.1 66.7 4.7 AD-800400.1 31.8 1.9 40.7 3.5 47.8 1.4 66.9 4.9 AD-800414.2 53.0 5.0 45.4 1.9 66.4 4.2 67.5 3.4 AD-801886.2 49.6 4.0 50.2 4.5 59.2 3.6 67.5 7.9 AD-961187.1 28.8 2.4 31.2 4.7 51.7 4.6 68.4 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9.0 60.3 13.3 107.8 12.8 AD-800974.2 62.8 3.6 69.2 4.8 80.5 5.3 108.5 6.8 AD-800850.2 68.8 7.6 64.0 1.7 83.0 5.1 110.0 9.7 AD-961066.1 64.2 6.4 68.6 9.0 94.8 8.0 110.6 11.9 AD-961220.1 91.6 11.9 97.9 17.5 106.8 19.0 111.3 15.8 AD-1010683.1 91.9 9.3 84.9 13.1 91.8 11.9 111.6 15.8 AD-961009.1 86.0 8.0 64.8 11.9 94.8 15.3 111.9 10.9 AD-961269.1 91.7 11.5 69.7 4.0 74.8 15.5 112.3 11.0 AD-961271.1 54.8 11.6 66.8 8.6 63.9 4.8 112.5 10.4 AD-961042.1 131.0 17.7 104.1 14.8 132.1 19.7 113.1 16.1 AD-961233.1 86.8 2.7 92.3 11.2 115.2 12.0 113.5 24.6 AD-1010691.1 83.2 13.4 70.3 12.5 70.5 20.0 114.3 9.2 AD-1010680.1 75.5 14.3 62.1 20.6 69.2 3.6 115.3 9.3 AD-997386.1 75.4 11.9 54.6 2.2 102.7 5.3 115.7 10.0 AD-801140.2 81.4 2.3 73.8 7.3 110.3 8.9 115.8 18.3 AD-996618.1 49.0 6.8 40.5 1.0 83.2 17.7 115.9 14.5 AD-1000451.1 102.3 7.4 100.7 10.4 117.9 11.6 116.1 6.2 AD-961024.1 102.3 1.9 90.5 8.4 129.4 9.6 116.2 7.7 AD-1010688.1 81.2 10.5 68.2 16.2 76.2 10.5 116.4 5.1 AD-999721.1 88.2 14.5 74.3 8.8 105.7 7.6 117.0 15.7 AD-999986.1 92.1 7.1 68.3 4.2 103.6 25.5 117.7 4.4 AD-961252.1 76.5 5.7 71.6 8.5 93.7 5.0 118.3 8.2 AD-1000133.1 108.6 9.9 104.8 4.5 103.4 14.8 118.4 22.2 AD-1010672.1 47.9 7.7 34.5 10.1 67.6 14.0 119.1 14.0 AD-961039.1 63.3 8.9 50.4 6.3 92.7 18.6 119.5 8.3 AD-800384.2 109.7 11.6 107.5 11.0 157.3 12.1 119.8 16.6 AD-998897.1 95.0 7.2 80.0 8.1 113.6 10.8 119.8 13.0 AD-996319.1 50.5 3.3 41.7 4.8 93.7 5.9 119.9 18.3 AD-996635.1 69.5 5.3 60.3 5.6 107.1 1.9 120.2 14.4 AD-1010678.1 88.1 12.5 85.5 10.6 89.0 17.2 121.1 15.0 AD-1010685.1 111.6 15.7 108.7 12.2 78.0 7.2 122.2 7.9 AD-961044.1 97.5 9.6 88.4 12.2 134.5 10.9 122.7 22.2 AD-1010686.1 78.7 6.5 76.7 7.0 86.1 26.9 123.1 19.5 AD-1010687.1 117.9 15.8 115.2 30.5 107.5 26.3 124.7 19.7 AD-994670.1 129.5 59.3 134.9 46.8 118.4 32.5 124.8 42.9 AD-996052.1 91.5 4.9 72.6 13.5 121.3 21.0 125.5 29.0 AD-961244.1 76.7 11.0 77.3 12.3 86.9 17.0 125.7 11.9 AD-1010689.1 97.6 12.4 70.6 13.0 72.1 13.8 125.7 12.1 AD-998894.1 99.2 7.1 92.6 8.1 127.6 21.1 126.2 23.1 AD-995824.1 79.3 6.1 79.4 5.6 132.8 21.0 127.8 17.2 AD-998346.1 63.5 10.6 50.5 11.1 83.0 29.0 127.9 17.0 AD-995660.1 136.0 20.1 113.3 16.3 138.8 21.7 128.3 27.5 AD-961204.1 131.2 6.3 120.0 23.3 93.0 6.9 128.4 16.1 AD-998261.1 92.0 5.8 69.2 3.7 120.4 14.9 128.8 10.2 AD-800975.2 68.4 14.1 70.0 14.9 93.4 23.3 128.9 30.2 AD-794914.1 54.4 20.5 45.0 8.5 85.8 5.5 129.1 22.5 AD-1010682.1 74.2 13.6 73.0 10.6 77.2 11.2 129.5 7.4 AD-961246.1 74.2 5.9 67.8 8.9 104.0 16.8 130.0 10.2 AD-961037.1 73.0 6.1 55.0 6.2 97.9 11.1 130.1 15.6 AD-961043.1 113.8 12.2 101.4 8.0 136.2 19.6 131.8 6.0 AD-798031.1 80.4 12.9 69.9 8.2 86.4 19.4 132.3 18.6 AD-961232.1 106.8 22.1 102.7 18.1 127.4 26.3 132.5 16.0 AD-996036.1 110.9 9.5 102.8 7.5 123.2 6.6 132.5 11.3 AD-995573.1 144.5 17.9 109.1 8.6 139.9 28.2 132.8 12.3 AD-995587.1 141.8 15.3 112.6 18.3 141.1 16.7 133.9 11.4 AD-961295.1 100.5 8.5 78.2 5.3 76.0 6.5 135.3 15.9 AD-999596.1 102.6 5.7 93.6 13.8 131.0 1.9 136.1 27.0 AD-996619.1 94.9 5.1 96.5 13.6 118.2 17.2 136.6 10.8 AD-795826.1 70.1 11.1 56.1 13.8 100.3 7.9 136.7 18.9 AD-998015.1 82.1 2.8 63.8 3.4 107.7 11.0 137.3 5.5 AD-1010675.1 90.0 16.1 76.8 13.1 99.9 24.3 137.7 24.0 AD-1010681.1 102.6 13.3 91.2 26.3 119.3 18.8 138.2 22.6 AD-995823.1 77.4 5.2 69.6 8.2 127.3 13.1 138.3 14.2 AD-999215.1 97.0 18.5 87.0 4.4 113.7 16.5 139.4 19.5 AD-801020.2 78.5 6.5 66.2 5.4 101.1 4.1 139.7 11.6 AD-999348.1 87.0 12.7 80.6 3.6 111.4 12.2 141.1 13.3 AD-996533.1 110.0 6.7 110.4 11.6 148.8 15.8 146.0 22.6 AD-961036.1 129.0 17.9 106.4 13.8 135.2 13.4 149.0 12.0 AD-961231.1 131.4 20.6 127.4 10.6 126.4 31.6 155.5 24.9 AD-997715.1 117.7 16.6 110.1 14.6 129.0 20.5 157.2 35.0 AD-801309.2 59.4 6.8 63.1 7.3 83.8 4.8 158.4 66.9

The results of the multi-dose screen in BE(2)-C cells expressing a SCN9A gene and treated with an exemplary set of SCN9A siRNAs is shown in Table 17 (correspond to siRNAs in Table 13A). The experiment was performed at a 0.1 nM, 1 nM, 10 nM, and 50 nM final duplex concentrations and the data are expressed as percent message remaining relative to non-targeting control.

Of the siRNA duplexes evaluated at 50 nM in Table 17, 5 achieved ≥90% knockdown of SCN9A, 52 achieved ≥80% knockdown of SCN9A, 180 achieved ≥60% knockdown of SCN9A, 254 achieved ≥30% knockdown of SCN9A, 261 achieved ≥20% knockdown of SCN9A, and 264 achieved ≥10% knockdown of SCN9A.

Of the siRNA duplexes evaluated at 10 nM in Table 17, 3 achieved ≥90% knockdown of SCN9A, 59 achieved ≥80% knockdown of SCN9A, 174 achieved ≥60% knockdown of SCN9A, 233 achieved ≥30% knockdown of SCN9A, 249 achieved ≥20% knockdown of SCN9A, and 255 achieved ≥10% knockdown of SCN9A.

Of the siRNA duplexes evaluated at 1 nM in Table 17, 2 achieved ≥90% knockdown of SCN9A, 15 achieved ≥80% knockdown of SCN9A, 109 achieved ≥60% knockdown of SCN9A, 228 achieved ≥30% knockdown of SCN9A, 247 achieved ≥20% knockdown of SCN9A, and 258 achieved ≥10% knockdown of SCN9A.

Of the siRNA duplexes evaluated at 0.1 nM in Table 17, 9 achieved ≥70% knockdown of SCN9A, 30 achieved ≥60% knockdown of SCN9A, 77 achieved ≥50% knockdown of SCN9A, 178 achieved ≥30% knockdown of SCN9A, 203 achieved ≥20% knockdown of SCN9A, and 225 achieved ≥10% knockdown of SCN9A.

TABLE 17 SCN9A in vitro multidose-dose screen with one set of exemplary human SCN9A siRNA duplexes (*the number following the decimal point in a duplex name merely refers to a batch production number) 50 nM 10 nM 1 nM 0.1 nM Mis- % message % message % message % message Duplex SC9NA match remaining St. Dev. remaining St. Dev. remaining St. Dev. remaining St. Dev. AD-1251302.1 HsSCN9A_ORF1rp 3 88.2 15.4 75.7 19.8 81.6 6.1 70.8 2.7 AD-1251303.1 HsSCN9A_ORF1rp 2 73.2 8.7 84.2 27.4 61.9 8.6 68.2 3.6 AD-1251304.1 HsSCN9A_ORF1rp 1 25.8 4.0 53.2 23.0 44.2 5.1 43.9 1.7 AD-1251305.1 HsSCN9A_ORF1rp 1 25.6 4.7 18.8 3.3 38.3 6.9 49.1 3.4 AD-1251306.1 HsSCN9A_ORF1rp 1 27.7 5.2 20.7 2.1 40.2 9.0 55.6 5.5 AD-1251307.1 HsSCN9A_ORF1rp 1 40.5 2.1 64.0 22.8 56.1 4.4 68.3 4.1 AD-1251315.1 HsSCN9A_ORF1rp 1 26.6 1.3 24.7 1.5 44.7 4.8 46.9 2.6 AD-1251310.1 HsSCN9A_ORF1rp 1 32.0 1.1 28.3 2.0 48.9 5.1 59.8 5.1 AD-961179.3 HsSCN9A_ORF1rp 1 33.9 2.9 29.3 3.2 42.5 4.8 49.9 3.7 AD-1251308.1 HsSCN9A_ORF1rp 1 40.3 3.7 29.4 2.8 49.1 6.4 58.1 3.5 AD-1251314.1 HsSCN9A_ORF1rp 1 41.7 3.0 34.1 2.9 58.0 8.3 58.8 3.4 AD-1251309.2 HsSCN9A_ORF1rp 1 48.4 3.9 34.4 1.1 66.1 10.1 60.3 4.9 AD-1251316.1 HsSCN9A_ORF1rp 1 43.4 2.5 37.1 6.7 49.8 1.7 72.8 16.2 AD-1251317.1 HsSCN9A_ORF1rp 1 27.2 2.9 58.7 43.5 34.3 2.0 40.3 2.7 AD-1251311.1 HsSCN9A_ORF1rp 1 45.9 14.8 58.9 37.2 59.2 4.8 69.3 24.8 AD-1251309.1 HsSCN9A_ORF1rp 1 53.5 3.2 80.0 46.2 57.9 4.1 64.7 5.8 AD-1251318.1 HsSCN9A_ORF1rp 1 23.3 1.3 18.0 2.7 30.2 5.3 37.7 7.8 AD-1251319.1 HsSCN9A_ORF1rp 1 25.4 0.9 21.3 1.9 48.0 13.8 46.8 1.6 AD-1251313.1 HsSCN9A_ORF1rp 1 36.3 1.6 30.8 7.3 53.0 4.0 65.6 4.2 AD-1251312.1 HsSCN9A_ORF1rp 1 54.4 6.9 38.8 2.4 66.9 5.4 69.3 3.5 AD-1251320.1 HsSCN9A_ORF1rp 1 26.5 2.0 43.2 29.5 39.8 5.2 47.7 5.3 AD-1251321.1 HsSCN9A_ORF1rp 0 19.3 3.3 15.3 3.5 38.1 12.7 36.2 2.4 AD-1251323.1 HsSCN9A_ORF1rp 0 17.3 4.1 18.9 9.8 27.2 4.7 35.9 5.5 AD-1251322.1 HsSCN9A_ORF1rp 1 16.3 2.4 22.5 10.0 27.5 1.2 40.8 5.4 AD-1251325.1 HsSCN9A_ORF1rp 1 19.6 2.8 30.6 15.0 29.2 5.3 35.4 1.6 AD-1251324.1 HsSCN9A_ORF1rp 1 22.0 9.0 33.4 14.4 27.5 6.9 33.2 2.7 AD-1251249.1 HsSCN9A_ORF1rp 1 25.0 2.2 15.0 1.3 29.6 6.1 36.6 5.4 AD-1251254.1 HsSCN9A_ORF1rp 1 19.3 1.3 17.6 2.9 30.4 4.1 43.8 20.0 AD-1251248.1 HsSCN9A_ORF1rp 1 29.8 5.2 17.8 2.4 35.8 4.6 47.0 6.4 AD-1251284.1 HsSCN9A_ORF1rp 1 21.8 1.9 19.0 2.4 30.1 3.3 32.2 1.8 AD-1251253.1 HsSCN9A_ORF1rp 1 28.6 2.0 22.0 3.0 48.2 5.9 102.4 47.0 AD-1251286.1 HsSCN9A_ORF1rp 1 25.4 4.1 22.6 3.5 36.5 5.9 37.3 3.7 AD-1251282.1 HsSCN9A_ORF1rp 1 30.9 8.8 22.8 9.3 36.9 5.5 50.8 3.4 AD-1010661.3 HsSCN9A_ORF1rp 1 34.2 4.6 23.1 5.5 48.8 11.5 56.1 6.3 AD-795305.3 HsSCN9A_ORF1rp 1 23.6 3.8 23.4 16.3 47.7 36.0 2.0 AD-1251250.1 HsSCN9A_ORF1rp 1 22.9 1.8 24.6 12.5 30.6 3.7 43.6 4.7 AD-1251283.1 HsSCN9A_ORF1rp 1 28.9 6.0 26.8 9.0 36.5 7.8 45.0 5.7 AD-1251281.1 HsSCN9A_ORF1rp 1 33.7 11.0 26.8 5.7 54.1 5.9 61.1 2.6 AD-1251255.1 HsSCN9A_ORF1rp 1 30.5 1.9 26.9 10.7 49.1 8.5 63.8 5.7 AD-1251289.1 HsSCN9A_ORF1rp 1 26.4 1.6 35.1 5.9 49.0 4.2 68.1 9.7 AD-1251252.1 HsSCN9A_ORF1rp 1 28.1 1.9 44.5 31.9 44.9 4.2 58.8 6.1 AD-1251285.1 HsSCN9A_ORF1rp 1 34.3 5.6 47.3 33.3 50.0 5.3 60.3 8.1 AD-1251291.1 HsSCN9A_ORF1rp 1 39.7 7.7 48.5 18.9 64.0 15.2 64.5 3.5 AD-1251290.1 HsSCN9A_ORF1rp 1 23.7 2.0 66.4 27.7 36.9 2.3 42.2 3.7 AD-1251251.1 HsSCN9A_ORF1rp 1 17.9 0.8 13.9 1.9 27.8 4.8 35.4 3.9 AD-1251287.1 HsSCN9A_ORF1rp 1 27.9 7.8 25.9 0.7 49.1 10.2 55.2 7.4 AD-1251288.1 HsSCN9A_ORF1rp 1 21.3 4.1 29.0 7.5 51.3 18.3 41.2 2.0 AD-1251326.1 HsSCN9A_ORF1rp 1 32.0 5.4 24.4 0.2 52.9 3.2 63.0 3.0 AD-1251327.1 HsSCN9A_ORF1rp 1 67.9 7.7 74.9 27.8 69.2 9.1 69.9 2.9 AD-1251328.1 HsSCN9A_ORF1rp 1 19.3 2.3 25.3 4.0 27.3 6.1 66.1 5.8 AD-1251329.1 HsSCN9A_ORF1rp 0 34.9 1.9 101.9 3.9 55.1 4.7 91.4 8.7 AD-1251330.1 HsSCN9A_ORF1rp 1 54.9 5.1 48.1 3.6 55.2 9.0 77.1 10.3 AD-795366.3 HsSCN9A_ORF1rp 1 18.6 5.7 15.7 2.4 26.1 8.5 42.8 7.0 AD-1251331.1 HsSCN9A_ORF1rp 1 17.6 2.0 20.9 1.5 30.8 6.1 56.5 4.6 AD-1251334.1 HsSCN9A_ORF1rp 1 20.1 5.8 26.4 2.8 24.2 2.8 37.1 6.4 AD-1251333.1 HsSCN9A_ORF1rp 1 27.0 1.6 28.8 4.2 40.8 4.7 59.1 6.6 AD-1251338.1 HsSCN9A_ORF1rp 1 28.5 2.0 31.6 6.3 47.9 0.7 92.7 10.2 AD-1251337.1 HsSCN9A_ORF1rp 1 39.3 2.5 41.6 5.2 65.7 13.5 98.8 6.2 AD-1251336.1 HsSCN9A_ORF1rp 1 45.9 3.6 54.4 6.2 72.8 7.3 106.0 11.0 AD-1251335.1 HsSCN9A_ORF1rp 1 52.5 6.1 71.4 5.5 66.2 6.9 101.9 22.0 AD-1251339.1 HsSCN9A_ORF1rp 1 25.4 1.0 28.3 2.1 51.9 2.0 78.1 7.9 AD-1251340.1 HsSCN9A_ORF1rp 1 34.1 3.9 29.5 5.2 45.8 1.2 65.6 8.5 AD-1251341.1 HsSCN9A_ORF1rp 1 51.9 2.9 48.3 5.0 51.3 3.3 60.1 7.4 AD-1251342.1 HsSCN9A_ORF1rp 1 18.3 4.3 20.8 1.3 20.2 2.5 27.9 2.1 AD-1251347.1 HsSCN9A_ORF1rp 1 25.4 4.3 15.9 5.6 34.8 4.4 55.2 6.2 AD-795371.3 HsSCN9A_ORF1rp 1 17.3 3.2 19.0 4.2 24.1 8.2 43.4 5.8 AD-1010663.3 HsSCN9A_ORF1rp 1 28.3 2.4 20.4 1.6 32.5 3.5 45.2 5.2 AD-1251301.1 HsSCN9A_ORF1rp 1 26.4 1.3 20.4 1.5 39.6 3.0 47.9 5.8 AD-1251348.1 HsSCN9A_ORF1rp 2 17.5 8.4 22.8 2.1 29.8 6.8 41.0 5.3 AD-1251343.1 HsSCN9A_ORF1rp 1 23.1 1.6 23.3 4.6 41.3 7.1 63.7 11.5 AD-1251346.1 HsSCN9A_ORF1rp 1 29.4 3.4 26.2 4.3 46.1 2.1 68.6 14.2 AD-1251299.1 HsSCN9A_ORF1rp 1 32.4 7.9 29.5 14.3 48.7 3.5 58.7 3.8 AD-1251345.1 HsSCN9A_ORF1rp 2 30.4 2.1 29.7 3.6 47.7 6.3 78.9 7.2 AD-1251349.1 HsSCN9A_ORF1rp 1 23.8 4.4 31.2 6.1 33.6 11.0 55.6 10.1 AD-1251292.1 HsSCN9A_ORF1rp 1 27.0 3.7 31.8 12.7 42.1 6.7 128.8 66.1 AD-1251293.1 HsSCN9A_ORF1rp 1 32.4 4.6 32.5 13.1 43.9 4.9 58.5 4.1 AD-1251294.1 HsSCN9A_ORF1rp 2 44.6 3.5 33.3 3.3 54.8 5.2 65.1 0.8 AD-1251344.1 HsSCN9A_ORF1rp 1 30.0 2.9 33.5 2.0 47.0 4.6 81.8 7.0 AD-1251300.1 HsSCN9A_ORF1rp 1 32.2 4.4 39.5 21.6 42.6 3.6 58.2 4.4 AD-1251295.1 HsSCN9A_ORF1rp 1 31.6 5.8 43.2 12.4 64.8 4.1 62.3 3.7 AD-1251296.1 HsSCN9A_ORF1rp 1 37.2 7.3 60.8 31.7 52.5 11.4 60.4 7.7 AD-1251350.1 HsSCN9A_ORF1rp 1 28.0 2.9 29.3 3.3 36.9 4.9 67.8 7.3 AD-1251351.1 HsSCN9A_ORF1rp 1 26.3 2.2 31.5 3.6 52.4 5.5 80.5 10.0 AD-1251353.1 HsSCN9A_ORF1rp 1 25.4 0.8 26.4 5.8 39.2 5.0 60.7 4.0 AD-1251352.1 HsSCN9A_ORF1rp 1 27.1 1.4 28.1 3.9 38.7 4.4 79.3 7.7 AD-1251298.1 HsSCN9A_ORF1rp 1 58.5 11.3 34.5 6.3 61.7 6.7 61.1 3.2 AD-1251297.1 HsSCN9A_ORF1rp 1 50.7 9.4 45.4 19.9 64.9 11.2 68.5 3.1 AD-1251354.1 HsSCN9A_ORF1rp 1 25.6 3.7 25.2 4.1 36.5 3.5 61.3 10.9 AD-1251355.1 HsSCN9A_ORF1rp 1 12.9 6.8 15.5 5.4 14.6 2.6 27.9 3.6 AD-1251356.1 HsSCN9A_ORF1rp 1 21.4 6.6 30.9 2.2 21.2 13.2 45.7 5.6 AD-1251357.1 HsSCN9A_ORF1rp 1 29.0 1.0 31.8 4.9 49.7 14.6 60.1 17.7 AD-1251358.1 HsSCN9A_ORF1rp 1 19.3 1.3 26.5 6.7 52.1 6.5 76.8 36.1 AD-1251359.1 HsSCN9A_ORF1rp 0 16.3 2.0 16.5 3.6 26.8 12.1 47.0 25.6 AD-1251360.1 HsSCN9A_ORF1rp 0 23.5 2.1 23.0 3.3 33.2 9.9 62.8 5.0 AD-1251361.1 HsSCN9A_ORF1rp 0 20.0 1.2 19.8 4.4 29.8 8.5 46.8 5.4 AD-1251363.1 HsSCN9A_ORF1rp 0 9.4 3.0 10.9 1.9 14.7 2.8 34.0 12.5 AD-1251362.1 HsSCN9A_ORF1rp 0 12.5 1.7 13.3 1.5 24.3 5.1 36.3 7.1 AD-1251364.1 HsSCN9A_ORF1rp 1 12.5 4.1 14.8 1.7 11.8 6.0 25.0 1.5 AD-1251372.1 HsSCN9A_ORF1rp 1 21.3 3.1 11.0 6.1 23.6 4.6 58.5 8.3 AD-1251366.1 HsSCN9A_ORF1rp 1 14.5 0.6 13.8 3.9 25.6 3.5 62.5 3.0 AD-1251367.1 HsSCN9A_ORF1rp 1 15.0 2.6 16.2 1.0 27.2 5.6 56.8 4.9 AD-795634.4 HsSCN9A_ORF1rp 1 14.1 0.7 16.8 1.9 28.4 7.4 64.4 2.7 AD-1251369.1 HsSCN9A_ORF1rp 2 19.5 0.8 17.4 2.7 20.9 6.9 41.7 11.7 AD-1251368.1 HsSCN9A_ORF1rp 1 17.8 1.5 19.2 2.7 33.2 2.1 50.7 2.7 AD-1251373.1 HsSCN9A_ORF1rp 1 25.3 1.3 22.3 2.8 30.5 15.1 42.5 22.0 AD-1251365.1 HsSCN9A_ORF1rp 1 17.8 1.2 22.4 2.2 28.4 4.6 49.9 11.6 AD-1251370.1 HsSCN9A_ORF1rp 1 26.1 3.8 24.4 3.3 10.0 2.8 49.2 12.9 AD-1251374.1 HsSCN9A_ORF1rp 1 23.6 4.5 27.5 6.8 20.9 9.6 69.2 12.6 AD-1251375.1 HsSCN9A_ORF1rp 0 22.3 1.0 21.8 9.4 30.5 14.9 63.2 2.8 AD-1251371.1 HsSCN9A_ORF1rp 1 29.4 2.4 27.1 2.4 25.0 6.3 65.7 8.7 AD-1251376.1 HsSCN9A_ORF1rp 1 15.5 2.3 14.2 4.8 16.8 4.7 27.4 7.1 AD-1251377.1 HsSCN9A_ORF1rp 1 10.7 6.8 15.6 2.6 10.1 3.8 22.6 5.2 AD-1251378.1 HsSCN9A_ORF1rp 1 20.9 1.8 21.1 3.7 10.0 5.3 48.6 19.4 AD-1251379.1 HsSCN9A_ORF1rp 1 39.1 4.1 42.8 3.2 53.8 8.1 96.8 2.1 AD-1251380.1 HsSCN9A_ORF1rp 0 22.4 3.5 21.1 1.1 22.0 4.9 50.0 2.7 AD-1251381.1 HsSCN9A_ORF1rp 0 22.0 1.9 22.1 2.9 32.0 8.8 51.8 2.8 AD-1251382.1 HsSCN9A_ORF1rp 1 25.7 2.1 20.8 4.3 40.1 15.4 76.5 9.5 AD-1251384.1 HsSCN9A_ORF1rp 1 15.0 0.8 11.8 4.4 20.7 1.8 40.7 1.4 AD-1251274.2 HsSCN9A_ORF1rp 1 22.3 1.4 14.2 5.0 29.3 2.4 38.5 4.7 AD-961188.3 HsSCN9A_ORF1rp 1 20.9 1.1 14.8 1.8 35.5 5.8 49.9 4.7 AD-1251383.1 HsSCN9A_ORF1rp 1 16.6 2.2 14.8 3.3 27.8 6.5 47.5 6.1 AD-1251269.1 HsSCN9A_ORF1rp 1 23.0 6.7 15.5 1.4 41.6 17.6 43.6 4.3 AD-1251270.1 HsSCN9A_ORF1rp 1 22.1 2.9 16.8 4.6 47.0 8.0 60.8 4.2 AD-1251268.1 HsSCN9A_ORF1rp 1 20.3 3.0 17.1 6.6 46.2 12.4 46.8 4.1 AD-1251274.1 HsSCN9A_ORF1rp 1 16.9 3.7 17.9 6.9 34.3 5.2 104.5 47.1 AD-1251271.1 HsSCN9A_ORF1rp 1 23.6 2.1 19.9 3.1 35.2 1.9 53.7 5.6 AD-1251275.2 HsSCN9A_ORF1rp 1 23.0 4.1 26.0 7.7 54.8 20.9 51.3 2.6 AD-1251275.1 HsSCN9A_ORF1rp 1 21.0 3.5 38.9 22.9 43.9 15.8 51.1 6.7 AD-1251385.1 HsSCN9A_ORF1rp 1 8.9 3.7 9.4 4.8 11.9 6.0 30.3 5.2 AD-1251272.1 HsSCN9A_ORF1rp 1 22.0 2.4 19.3 6.8 31.8 2.2 54.8 4.8 AD-1251386.1 HsSCN9A_ORF1rp 0 24.8 2.7 26.0 2.1 27.1 13.8 47.2 17.2 AD-1251273.1 HsSCN9A_ORF1rp 1 24.4 1.5 50.5 19.9 35.7 7.0 49.2 1.9 AD-1251390.1 HsSCN9A_ORF1rp 1 23.4 1.8 13.0 4.8 37.9 11.8 59.5 9.9 AD-1251398.1 HsSCN9A_ORF1rp 1 19.9 10.8 16.2 3.1 14.6 6.5 47.5 8.8 AD-1251396.1 HsSCN9A_ORF1rp 1 23.8 1.5 17.1 4.6 31.1 8.8 55.7 3.6 AD-1251399.1 HsSCN9A_ORF1rp 1 20.2 4.6 17.6 3.9 19.9 6.1 39.4 17.9 AD-795913.3 HsSCN9A_ORF1rp 1 19.2 1.0 18.1 2.2 31.3 8.2 62.2 3.8 AD-1251400.1 HsSCN9A_ORF1rp 1 22.4 3.4 19.1 2.1 29.6 14.6 58.7 5.9 AD-1251388.1 HsSCN9A_ORF1rp 1 21.1 1.8 21.5 2.8 33.9 13.3 62.8 6.4 AD-1251397.1 HsSCN9A_ORF1rp 1 30.5 2.0 24.4 12.3 51.6 11.2 63.9 12.3 AD-1251395.1 HsSCN9A_ORF1rp 1 29.4 4.9 26.1 5.4 40.4 12.2 66.6 8.5 AD-1251387.1 HsSCN9A_ORF1rp 1 27.9 1.4 28.4 3.1 43.0 18.2 80.9 9.0 AD-1251389.1 HsSCN9A_ORF1rp 1 37.3 2.3 35.9 9.6 60.0 7.4 87.0 15.3 AD-1251393.1 HsSCN9A_ORF1rp 1 39.6 2.8 41.8 6.2 60.0 21.7 105.3 8.9 AD-1251394.1 HsSCN9A_ORF1rp 1 50.4 2.6 42.5 11.2 83.4 17.4 98.1 10.9 AD-1251401.1 HsSCN9A_ORF1rp 1 59.1 3.9 46.0 9.7 51.5 22.8 77.7 15.2 AD-1251391.1 HsSCN9A_ORF1rp 1 10.1 6.8 16.4 8.0 15.4 3.1 47.9 20.6 AD-1251392.1 HsSCN9A_ORF1rp 1 22.9 2.8 20.8 5.9 21.1 5.7 71.0 16.2 AD-1251402.1 HsSCN9A_ORF1rp 1 45.1 7.4 40.7 6.9 87.2 6.8 86.2 10.2 AD-1251403.1 HsSCN9A_ORF1rp 1 35.1 2.3 31.3 3.4 47.0 8.7 62.7 3.4 AD-1251404.1 HsSCN9A_ORF1rp 1 55.3 6.4 58.0 15.5 48.7 18.2 73.5 8.5 AD-1251405.1 HsSCN9A_ORF1rp 1 22.8 2.4 25.5 5.8 25.1 12.5 37.4 19.6 AD-1251406.1 HsSCN9A_ORF2rp 0 29.5 11.4 34.2 5.4 26.3 4.9 38.7 13.3 AD-1251407.1 HsSCN9A_ORF2rp 1 26.6 2.6 26.2 2.1 43.8 8.8 63.4 16.8 AD-1251408.1 HsSCN9A_ORF2rp 1 14.1 4.2 17.4 4.7 22.3 6.1 54.1 14.5 AD-1251409.1 HsSCN9A_ORF2rp 0 17.7 2.5 17.5 5.5 28.9 7.3 55.6 9.0 AD-1251411.1 HsSCN9A_ORF2rp 1 11.9 0.5 11.5 2.5 16.2 2.1 29.2 3.6 AD-1251410.1 HsSCN9A_ORF2rp 1 12.0 2.1 11.7 2.9 17.7 2.5 29.6 7.2 AD-1251412.1 HsSCN9A_ORF2rp 1 4.7 1.7 7.6 3.0 15.2 3.4 23.1 9.5 AD-796825.3 HsSCN9A_ORF2rp 1 5.2 2.5 9.6 2.6 16.6 3.1 35.0 7.7 AD-1251413.1 HsSCN9A_ORF2rp 1 14.2 2.9 12.0 1.9 26.6 7.3 46.9 4.2 AD-1251414.1 HsSCN9A_ORF2rp 1 13.6 1.7 15.4 2.7 31.7 4.6 64.8 9.0 AD-1251415.1 HsSCN9A_ORF2rp 1 13.5 0.7 15.9 2.4 23.0 3.6 51.2 6.1 AD-1251416.1 HsSCN9A_ORF2rp 1 12.8 2.2 17.3 2.6 31.1 2.8 57.0 6.5 AD-1251417.1 HsSCN9A_ORF2rp 0 11.3 2.4 13.5 3.9 23.6 2.2 42.4 10.3 AD-1251418.1 HsSCN9A_ORF2rp 1 46.4 5.0 48.1 2.6 50.6 2.9 57.6 9.0 AD-1251419.1 HsSCN9A_ORF2rp 1 8.7 1.3 12.9 1.4 23.0 2.3 27.2 3.2 AD-1251420.1 HsSCN9A_ORF2rp 1 15.3 1.1 18.7 1.3 35.5 2.9 44.8 3.1 AD-1251421.1 HsSCN9A_ORF2rp 1 16.5 1.2 18.2 2.7 32.3 4.0 56.2 6.1 AD-1251422.1 HsSCN9A_ORF2rp 1 21.9 2.5 23.5 4.6 36.5 3.7 68.5 4.2 AD-1251423.1 HsSCN9A_ORF2rp 1 48.6 3.7 45.7 3.8 62.7 3.8 84.1 8.5 AD-1251425.1 HsSCN9A_ORF2rp 0 18.0 2.3 25.7 3.4 28.3 2.9 45.7 2.3 AD-1251427.1 HsSCN9A_ORF2rp 1 18.5 2.3 22.0 1.3 29.6 1.3 42.5 3.1 AD-1251426.1 HsSCN9A_ORF2rp 1 29.7 1.9 29.7 3.0 38.7 3.9 67.0 10.6 AD-1251428.1 HsSCN9A_ORF2rp 1 12.5 0.8 17.5 1.9 23.6 0.1 30.9 3.4 AD-797564.4 HsSCN9A_ORF2rp 1 21.2 3.5 23.9 6.1 39.4 2.3 69.2 7.9 AD-1251434.1 HsSCN9A_ORF2rp 1 16.3 1.0 25.1 4.2 34.4 3.1 39.1 2.5 AD-1251431.1 HsSCN9A_ORF2rp 2 21.3 3.5 27.4 5.5 34.6 5.4 62.8 3.1 AD-1251433.1 HsSCN9A_ORF2rp 1 22.1 6.3 28.4 2.7 39.3 2.2 48.6 7.5 AD-1251430.1 HsSCN9A_ORF2rp 1 32.1 3.3 30.9 4.2 49.6 7.5 81.5 6.4 AD-1251429.1 HsSCN9A_ORF2rp 1 30.0 4.6 33.3 9.9 52.0 3.4 92.1 4.8 AD-1251435.1 HsSCN9A_ORF2rp 1 34.9 6.3 47.8 8.5 58.1 7.4 93.2 14.2 AD-1251438.1 HsSCN9A_ORF2rp 1 20.1 3.8 25.1 5.2 35.5 3.8 55.5 2.9 AD-1251436.1 HsSCN9A_ORF2rp 1 25.0 3.0 33.4 11.1 47.7 9.8 86.2 24.0 AD-1251437.1 HsSCN9A_ORF2rp 1 24.6 3.9 34.4 3.9 41.8 7.1 65.3 7.3 AD-797565.4 HsSCN9A_ORF2rp 1 28.2 6.0 39.3 8.0 54.4 0.0 76.1 7.2 AD-1251443.1 HsSCN9A_ORF2rp 1 43.2 4.4 52.4 3.6 63.0 4.0 94.7 10.5 AD-1251444.1 HsSCN9A_ORF2rp 1 39.3 7.9 54.5 13.1 59.8 14.9 72.2 9.9 AD-1251442.1 HsSCN9A_ORF2rp 1 44.2 5.8 61.1 9.7 76.8 8.9 102.9 5.6 AD-1251441.1 HsSCN9A_ORF2rp 1 51.0 10.9 71.0 14.2 78.9 12.0 119.7 14.5 AD-1251445.1 HsSCN9A_ORF2rp 0 56.7 7.7 57.9 1.8 84.7 14.2 101.1 18.9 AD-1251439.1 HsSCN9A_ORF2rp 1 21.7 4.1 30.7 2.6 35.2 0.3 49.0 4.9 AD-1251447.1 HsSCN9A_ORF2rp 0 34.1 3.4 32.8 4.6 47.4 1.8 77.8 6.3 AD-1251446.1 HsSCN9A_ORF2rp 0 29.8 4.3 34.6 0.9 46.9 9.3 64.0 8.0 AD-1251448.1 HsSCN9A_ORF2rp 1 21.7 3.3 24.6 3.4 36.2 2.3 57.2 8.1 AD-1251450.1 HsSCN9A_ORF2rp 1 45.4 5.5 64.0 8.0 80.5 11.1 90.5 5.5 AD-1251449.1 HsSCN9A_ORF2rp 1 39.5 1.9 66.5 8.8 70.0 13.7 79.7 8.7 AD-1251451.1 HsSCN9A_ORF2rp 1 151.3 26.1 173.5 16.6 134.6 12.6 112.4 20.9 AD-1251453.1 HsSCN9A_3UTR2 1 63.7 11.4 79.2 7.3 88.9 12.9 94.1 6.4 AD-1251452.1 HsSCN9A_3UTR2 1 71.3 4.8 81.2 9.3 85.0 10.8 82.6 5.4 AD-1251454.1 HsSCN9A_3UTR2 1 63.5 13.3 79.3 9.2 75.8 9.3 97.6 6.8 AD-1251455.1 HsSCN9A_3UTR2 1 51.7 9.1 46.1 4.9 63.5 3.0 80.6 2.1 AD-1251456.1 HsSCN9A_3UTR2 1 64.3 6.0 90.5 11.6 78.4 4.2 101.9 20.7 AD-1251457.1 HsSCN9A_3UTR2 1 79.1 14.7 110.0 9.9 115.5 20.4 112.2 12.8 AD-1251459.1 HsSCN9A_3UTR2 1 67.9 17.2 95.3 3.1 98.0 8.5 98.4 13.2 AD-1251458.1 HsSCN9A_3UTR2 1 65.7 8.9 96.7 5.2 89.7 8.1 92.6 19.5 AD-1251462.1 HsSCN9A_3UTR2 0 53.7 11.0 45.4 8.0 69.1 7.1 92.5 10.8 AD-1251461.1 HsSCN9A_3UTR2 0 39.5 4.4 52.2 9.5 52.4 10.0 73.4 7.2 AD-1251468.1 HsSCN9A_3UTR2 0 57.2 11.7 69.7 6.9 69.9 27.8 96.3 13.0 AD-1251463.1 HsSCN9A_3UTR2 0 59.0 6.0 71.0 7.3 76.1 25.4 84.5 0.7 AD-1251460.1 HsSCN9A_3UTR2 0 51.7 11.9 76.8 8.3 73.0 13.5 78.6 10.6 AD-1251469.1 HsSCN9A_3UTR2 0 58.2 6.4 80.9 6.3 86.4 9.0 93.7 7.8 AD-801647.3 HsSCN9A_3UTR2 0 64.5 11.9 83.8 8.1 78.4 5.2 80.1 7.9 AD-1251467.1 HsSCN9A_3UTR2 0 56.8 15.2 100.9 15.2 104.8 8.8 86.3 15.7 AD-1251466.1 HsSCN9A_3UTR2 0 72.3 18.5 105.2 10.0 112.2 10.3 113.0 13.4 AD-1251470.1 HsSCN9A_3UTR2 1 44.9 7.9 52.3 9.7 61.4 3.7 63.6 4.1 AD-1251471.1 HsSCN9A_3UTR2 1 51.3 2.7 69.5 5.1 54.2 3.3 80.1 11.2 AD-1251465.1 HsSCN9A_3UTR2 0 66.1 11.7 76.8 7.3 84.5 9.8 94.9 20.2 AD-1251472.1 HsSCN9A_3UTR2 1 86.2 4.2 98.3 12.9 76.8 8.6 109.5 6.7 AD-1251464.1 HsSCN9A_3UTR2 0 77.4 9.8 108.6 13.9 115.7 4.2 106.4 8.1 AD-1251473.1 HsSCN9A_3UTR2 0 57.3 1.8 68.9 6.8 76.5 6.6 84.0 8.0 AD-1251474.1 HsSCN9A_3UTR2 0 57.4 9.3 74.5 4.4 71.6 7.7 84.8 1.6 AD-1251475.1 HsSCN9A_3UTR2 1 57.3 6.3 64.7 8.7 70.6 9.1 80.0 9.0 AD-1251476.1 HsSCN9A_3UTR2 1 55.1 8.6 88.3 7.1 79.4 6.6 90.9 18.5 AD-1251279.1 HsSCN9A_3UTR2 0 41.1 2.9 30.9 2.1 38.7 4.8 46.6 2.3 AD-1251276.1 HsSCN9A_3UTR2 0 39.0 7.0 33.4 11.2 49.1 7.2 54.6 8.2 AD-1251280.1 HsSCN9A_3UTR2 0 43.9 2.9 41.1 13.8 43.0 4.2 46.3 3.9 AD-1251277.1 HsSCN9A_3UTR2 0 42.6 6.4 47.0 24.3 51.8 8.4 52.3 4.9 AD-961334.3 HsSCN9A_3UTR2 0 52.6 8.4 68.4 20.0 52.5 7.6 59.3 5.8 AD-1251278.1 HsSCN9A_3UTR2 0 36.6 3.1 45.6 20.8 44.7 4.7 47.2 2.1 AD-1251477.1 HsSCN9A_3UTR2 1 46.1 17.5 58.6 8.4 90.9 1.7 93.4 12.8 AD-1251478.1 HsSCN9A_3UTR2 1 78.6 3.2 82.8 1.1 77.7 8.7 112.7 15.5 AD-1251479.1 HsSCN9A_3UTR2 0 81.1 10.6 103.1 8.9 91.1 9.8 115.5 10.6 AD-1251481.1 HsSCN9A_3UTR2 1 65.0 4.7 76.1 9.1 73.4 3.7 98.6 9.3 AD-1251480.1 HsSCN9A_3UTR2 1 69.5 4.9 76.6 2.6 88.1 8.3 117.5 12.9 AD-1251482.1 HsSCN9A_3UTR2 1 60.8 2.9 65.6 13.3 72.6 8.4 91.2 14.0 AD-1251483.1 HsSCN9A_3UTR2 1 62.5 12.5 75.6 7.9 75.4 6.0 91.5 12.7 AD-1251492.1 HsSCN9A_3UTR2 0 22.3 2.6 29.1 3.3 33.3 3.4 52.8 7.6 AD-1251485.1 HsSCN9A_3UTR2 0 27.9 3.7 33.9 7.3 41.4 5.4 56.2 6.4 AD-802471.4 HsSCN9A_3UTR2 0 38.5 4.6 39.0 5.3 57.9 8.0 82.7 17.2 AD-1251486.1 HsSCN9A_3UTR2 0 38.5 2.8 39.8 8.1 51.6 4.4 78.1 11.1 AD-1251484.1 HsSCN9A_3UTR2 0 33.7 2.8 39.9 9.1 72.9 27.3 76.1 3.9 AD-1251491.1 HsSCN9A_3UTR2 0 33.7 7.5 45.6 5.7 48.4 6.8 61.9 11.3 AD-1251487.1 HsSCN9A_3UTR2 0 46.0 7.1 50.1 12.4 61.6 10.6 82.9 8.4 AD-1251488.1 HsSCN9A_3UTR2 0 47.9 6.4 52.1 6.6 57.2 8.0 82.0 7.7 AD-1251490.1 HsSCN9A_3UTR2 0 46.5 12.0 53.9 17.4 52.6 4.1 79.0 5.3 AD-1251494.1 HsSCN9A_3UTR2 1 51.7 6.1 42.6 2.8 51.8 7.3 80.5 5.4 AD-1251493.1 HsSCN9A_3UTR2 0 28.2 1.2 30.4 2.3 38.7 3.8 53.6 2.8 AD-1251489.1 HsSCN9A_3UTR2 0 38.9 4.4 54.3 8.1 59.3 13.6 83.7 16.5 AD-1251495.1 HsSCN9A_3UTR2 1 67.8 6.5 58.8 3.1 68.8 14.7 77.6 25.9 AD-1251496.1 HsSCN9A_3UTR2 1 61.6 7.8 48.6 5.8 64.2 7.1 78.7 7.9 AD-1251497.1 HsSCN9A_3UTR2 1 78.9 10.4 71.3 9.4 67.7 5.5 93.1 6.0 AD-1251498.1 HsSCN9A_3UTR2 1 91.0 6.2 79.9 14.1 79.0 11.5 89.1 10.4 AD-802552.3 HsSCN9A_3UTR2 0 45.8 3.9 31.8 4.5 60.8 9.9 51.1 3.4 AD-1251267.1 HsSCN9A_3UTR2 0 44.9 2.7 32.2 2.1 48.4 2.2 55.7 3.8 AD-1251260.1 HsSCN9A_3UTR2 0 48.1 7.6 33.0 6.6 59.5 10.8 58.4 1.8 AD-1251256.1 HsSCN9A_3UTR2 0 42.3 4.0 33.6 8.3 47.5 3.2 52.8 3.1 AD-1251265.1 HsSCN9A_3UTR2 0 50.9 6.8 34.5 10.1 60.0 16.7 58.4 4.3 AD-1251257.1 HsSCN9A_3UTR2 0 50.0 5.2 40.8 7.9 50.1 4.1 65.8 11.6 AD-1251266.1 HsSCN9A_3UTR2 0 52.4 7.1 41.8 6.8 58.0 5.5 70.0 16.1 AD-1251264.1 HsSCN9A_3UTR2 1 49.2 3.0 47.6 11.3 64.5 5.4 71.7 10.0 AD-1251259.1 HsSCN9A_3UTR2 0 49.0 4.7 48.9 23.0 49.1 3.1 64.4 1.8 AD-1251258.1 HsSCN9A_3UTR2 0 47.3 8.8 54.9 42.4 52.1 7.1 52.7 4.2 AD-1251263.1 HsSCN9A_3UTR2 0 30.4 1.6 22.8 4.0 67.4 15.5 71.5 24.2 AD-1251262.1 HsSCN9A_3UTR2 0 45.1 3.6 30.5 4.3 57.8 3.6 57.4 1.7 AD-1251261.1 HsSCN9A_3UTR2 0 43.1 5.3 34.1 10.0 47.5 5.0 51.5 4.8

Example 4. In Vivo Screening of SCN9A siRNA

Experimental Methods

Wildtype B6/C57 mice (Charles Rivers Laboratory) were retro-orbitally injected with human SCN9A constructs designed to span various regions of human SCN9A (e.g., the 3′ UTR_AAV1 (positions 6266 to 7998), 3′UTR-AAV2 (positions 7999 to 9750) and two open reading frames (ORF-1 (positions 299 to 2441) or ORF-2 (positions 2392 to 4354)) packaged in AAV particles (2×10¹⁰ gc/mouse). After two weeks, mice were injected subcutaneously with 3 mg/kg of exemplary siRNAs (C16, VCP, or GalNAc) (Tables 4A, 5A, 6A, 18 (also summarized in FIGS. 1A-1C) or 20 (also summarized in FIGS. 3A-3D), or a PBS or non-targeting siRNA control (Table 9). On day 14 post-treatment, livers were harvested for qPCR analysis with a probe specifically recognizing SCN9A. Mouse GAPDH was used as normalization control. Relative levels of SCN9A mRNA in the liver were calculated with the delta/delta Ct method, normalized to the control groups, and is depicted as the percent message remaining in Tables 10-12, 19, and 21 below.

TABLE 9 Control siRNA Sequences Duplex Seq ID No: Modified Seq ID No: Unmodified Name Target Strand (modified) Sequences (unmodified) Sequences AD- none Sense 3695 asascaguGfuUfCfUf 3696 AACAGUGUUC 64228.39 ugcucuauaaL96 UUGCUCUAUA A AD- mTTR Antisense 3697 usUfsauaGfaGfCfaa 3698 UUAUAGAGCA 86460 gaAfcAfcuguususu AGAACACUGU UUU

TABLE 18 Exemplary SCN9A duplexes investigated and corresponding chemistry that target ORF-1 of SCN9A (e.g., positions 299-2441). In this table the column “Duplex Name” provides the numerical part of the duplex name with a suffix (number following the decimal point in a duplex name) that merely refers to a batch production number. The suffix can be omitted from the duplex name without changing the chemical structure. SEQ ID Duplex Name Strand NO: Modified Sequence AD-795305.2 sense 5330 usgsucg(Ahd)GfuAfCfAfcuuuuacugaL96 (parent) anti- 5346 VPusCfsaguAfaAfAfguguAfcUfcgacasusu sense AD-1251249.1 sense 5331 usgsucgaguAfCfAfcuuu(Uhd)acugaL96 anti- 5347 VPusCfsagdTadAaaguguAfcUfcgacasusu sense AD-1251251.1 sense 5332 uscsgaguAfCfAfcuuu(Uhd)acugaL96 anti- 5348 VPusCfsagdTadAaaguguAfcUfcgascsg sense AD-1010663.2 sense 5333 usgsuag(Ghd)agdAadTucacuuuucaL96 (parent) anti- 5349 VPusdGsaadAadGugaadTudCudCcuacascsa sense AD-1251301.1 sense 5334 usgsuaggagdAaUfUfcac(Uhd)uuucaL96 anti- 5350 VPudGaadAa(G2p)ugaadTudCudCcuacascsg sense AD-961179.3 sense 5335 asasggg(Ahd)aadAcdAaucuuccguaL96 (parent) anti- 5351 VPusdAscgdGadAgauudGudTudTcccuususg sense AD-1251317.1 sense 5336 asasgggaaaAfCfAfaucu(Uhd)ccguaL96 anti- 5352 VPudAcgdGa(A2p)gauudGuUfudTcccuususg sense AD-1251318.1 sense 5337 asgsggaaAfaCfAfAfucuu(Chd)cguuaL96 anti- 5353 VPusAfsacdGgdAagauugUfuUfucccususu sense AD-1251323.1 sense 5338 gsasaaa(Chd)aaUfCfUfuccguuucaaL96 anti- 5354 VPuUfgadAa(C2p)ggaagaUfudGuuuucscsc sense AD-1251325.1 sense 5339 asasaacaauCfUfUfccgu(Uhd)ucaaaL96 anti- 5355 VPuUfugdAadAcggadAgdAuUfguuuuscsc sense AD-795634.3 sense 5340 asgscau(Ahd)AfaUfGfUfuuucgaaauaL96 (parent) anti- 5356 VPusAfsuuuCfgAfAfaacaUfuUfaugcususc sense AD-1251363.1 sense 5341 gsasagcauadAaUfguuu(Uhd)cgaaaL96 anti- 5357 VPuUfucdGadAaacadTuUfaUfgcuucsasg sense AD-1251364.1 sense 5342 asasgca(Uhd)aadAudGuuuucgaaaaL96 anti- 5358 VPuUfuudCgdAaaacdAuUfudAugcuuscsg sense AD-1251373.1 sense 5343 asgscauaaaUfgUfuuu(Chd)gaaauaL96 anti- 5359 VPudAuudTc(G2p)aaaadCaUfuUfaugcuscsc sense AD-1251385.1 sense 5344 asusgau(Chd)UfuCfUfUfugucguaguaL96 (parent: AD- anti- 5360 VPudAcudAcdGacaadAgdAadGaucausgsu 795913) sense AD-1251391.1 sense 5345 uscsu(Uhd)CfuUfudGucguagugaaL96 (parent: AD- anti- 5361 VPusUfscadCu(Agn)cgacdAaAfgAfagasusc 795913) sense

TABLE 20 Exemplary SCN9A duplexes investigated and corresponding chemistry that target region 2 of the 3′ UTR of SCN9A (HsSCN9A_3UTR2, e.g., positions 7999 to 9750), ORF-1 of SCN9A (HsSCN9A_ORF1rp, e.g., positions 299-2441), and ORF2 of SCN9A (HsSCN9A_ORF2rp, e.g., positions 2392-4345). In this table the column “Duplex Name” provides the numerical part of the duplex name with a suffix (number following the decimal point in a duplex name) production number. The suffix can be omitted from the duplex name without changing the that merely refers to a batch chemical structure. Duplex SEQ ID Parent AAV Name Strand NO: Modified Sequence HsSCN9A_ AD- sense 5410 csasagugUfuCfCfUfacug(Uhd) AD- 3UTR2 1251492.2 caugaL96 802471 anti- 5426 VPuCfaudGa(C2p)aguaggAfaC sense facuugscsc AD- sense 5411 csasaca(Chd)aadTudTcuucuua Self 961334.2 gcaL96 (Parent) anti- 5427 VPusdGscudAadGaagadAadTu sense dGuguugsusu AD- sense 5412 csasaca(Chd)aaufUfUfcuucuu AD- 1251279.2 agcaL96 961334 anti- 5428 VPudGcudAadGaagadAaufud sense Guguugsusu HsSCN9A_ AD- sense 5413 usgsucgaguAfCfAfcuuu(Uhd)a AD- ORF1rp 1251284.2 cugaL96 1010661 anti- 5429 VPusCfsagdTadAaagudGuAfcd sense Tcgacasusu AD- sense 5414 ususcug(Uhd)guAfgdGagaauu AD- 1251334.2 cacaL96 795366 anti- 5430 VPusdGsugdAa(U2p)ucucdCu sense AfcAfcagaasgsc AD- sense 5415 asusaaa(Uhd)guUfUfUfcgaaau AD- 1251377.2 ucaaL96 795634 anti- 5431 VPusufsgadAudTucgaaaAfcAf sense uuuausgsu AD- sense 5416 gsasucu(Uhd)CfuUfudGucgua AD- 1251398.2 gugaaL96 795913 anti- 5432 VPuufcadCu(A2p)cgacdAaAfg sense Afagaucsgsu AD- sense 5417 gsasucu(Uhd)CfuUfudGUfcgu AD- 1251399.2 agugaaL96 795913 anti- 5433 VPuufcadCu(A2p)cgacdAaAfg sense Afagaucsgsu AD- sense 5418 csasuga(Uhd)cudTcdTuugucgu Self 961188.2 agaL96 (Parent) anti- 5434 VPusdCsuadCgdAcaaadGadAg sense dAucaugsusa AD- sense 5419 csasuga(Uhd)cuufCfUfuugucg AD- 1251274.3 uagaL96 961188 anti- 5435 VPuCfuadCgdAcaaadGadAgdA sense ucaugsusg HsSCN9A_ AD- sense 5420 ususugu(Ahd)GfaufCfUfugcaa Self ORF2rp 796825.2 uuacaL96 (Parent) anti- 5436 VPusGfsuaaUfuGfCfaagaUfcUf sense acaaasasg AD- sense 5421 ususuug(Uhd)agAfUfCfuugcaa AD- 1251411.2 uuaaL96 796825 anti- 5437 VPusufsaadTu(G2p)caagauCfu sense Afcaaagscsc AD- sense 5422 gsusaga(Uhd)CfuufgCfaauuac AD- 1251419.2 cauaL96 796825 anti- 5438 VPudAugdGudAauugdCaAfgAf sense ucuacsgsg AD- sense 5423 usasugu(Ghd)AfaAfCfAfaaccu Self 797564.3 uacgaL96 (Parent) anti- 5439 VPusCfsguaAfgGfUfuuguUfuCf sense acauasasu AD- sense 5424 ususaug(Uhd)gaAfAfCfaaaccu AD- 1251428.2 uacaL96 1251428 anti- 5440 VPudGuadAg(G2p)uuuguuufc sense Afcauaasusu AD- sense 5425 usasugugAfaAfCfAfaacc(Uhd) AD- 1251434.2 uacgaL96 1251428 anti- 5441 VPuCfgudAa(G2p)guuuguUfu sense Cfacauasgsu

Results

Table 10 (siRNA duplexes correspond to siRNA sequences in Tables 4A and 5A) demonstrates the results of the in vivo screen for the ORF-1 targeting duplexes, and includes siRNA duplexes with Fluoro and Non-Fluoro chemistries. Of the siRNA duplexes evaluated in the in vivo screen shown in Table 10, 1 achieved ≥80% knockdown of SCN9A, 8 achieved ≥60% knockdown of SCN9A, 13 achieved ≥40% knockdown of SCN9A, and 15 achieved ≥20% knockdown of SCN9A.

TABLE 10 Efficacy and duration of exemplary ORF-1 targeting SCN9A siRNAs in mice % message remaining at Treatment* Chemistry day 14 post-treatment St. Dev. PBS N/A 100.00 19.46 Naïve (AAV-only) 115.79 22.37 AD-64228.39 (AAV- Fluoro 59.06 27.45 control) AD-961179.2 NonFluoro 20.49 0.95 AD-795305.2 Fluoro 21.96 5.16 AD-1010661.2 NonFluoro 41.08 6.19 AD-795366.2 Fluoro 29.15 4.74 AD-1010662.2 NonFluoro 52.17 4.57 AD-795371.2 Fluoro 16.30 6.34 AD-1010663.2 NonFluoro 21.60 1.20 AD-795634.3 Fluoro 20.80 6.50 AD-795739.2 Fluoro 41.18 10.45 AD-1010664.2 NonFluoro 71.93 4.59 AD-961188.2 NonFluoro 38.51 0.36 AD-961189.2 NonFluoro 55.25 14.04 AD-795913.2 Fluoro 26.74 5.61 AD-795914.2 Fluoro 76.20 11.80 AD-961192.2 NonFluoro 104.26 12.37 AD-1010671.2 NonFluoro 98.29 17.97 AD-796618.2 Fluoro 57.42 1.30 (*the number following the decimal point in a duplex name merely refers to a batch production number)

Table 11 (siRNA duplexes correspond to siRNA sequences in Tables 4A, 5A, and 6A) demonstrates the results of the in vivo screen for the ORF-2-targeting duplexes as well as the 3′UTR_AAV1 and 3′UTR_AAV2 targeting duplexes and includes siRNA duplexes with Fluoro, Non-Fluoro, Fluoro+GNA chemistries. Of the ORF-2 targeting siRNA duplexes evaluated in the in vivo screen shown in Table 11, 3 achieved ≥80% knockdown of SCN9A, 4 achieved ≥30% knockdown of SCN9A, and 5 achieved ≥20% knockdown of SCN9A. Of the 3′UTR_AAV1 targeting siRNA duplexes (positions 6266 to 7998) evaluated in this screen shown in Table 11, 2 achieved ≥20% knockdown of SCN9A. Of the 3′UTR_AAV2 targeting siRNA duplexes (positions 7999 to 9750) evaluated in this screen shown in Table 11, 2 achieved ≥60% knockdown of SCN9A, and 5 achieved ≥30% knockdown of SCN9A.

TABLE 11 Efficacy and duration of exemplary ORF-2 and 3′UTR targeting SCN9A siRNAs in mice % message remaining at day 14 post- St. AAV Treatment* Chemistry treatment Dev. HsSCN9A_ORF2rp PBS n/a 100.00 13.04 naïve (AAV-only) 96.31 19.26 AD-796825.1 Fluoro 12.49 2.90 AD-961207.1 NonFluoro 71.01 17.81 AD-961208.1 NonFluoro 66.66 10.33 AD-797564.2 Fluoro 13.59 5.19 AD-797565.2 Fluoro 12.26 1.86 3′UTR_AAV1 PBS n/a 100.00 21.53 naïve (AAV-only) 117.95 19.80 AD-800819.1 Fluoro 79.02 5.13 AD-1010693.1 NonFluoro 70.89 9.77 3′UTR_AAV2 PBS n/a 100.00 28.24 naïve (AAV-only) 114.62 35.06 AD-802503.1 Fluoro 50.95 7.80 AD-802552.1 Fluoro 31.59 5.19 AD-1002101.1 Fluoro + 55.04 23.31 GNA AD-802625.2 Fluoro 47.78 4.47 AD-802853.2 Fluoro 35.44 5.41 (*the number following the decimal point in a duplex name merely refers to a batch production number)

Table 12 (siRNA duplexes correspond to siRNA sequences in Tables 4A, 5A, and 6A) demonstrates the results of the in vivo screen for 3′UTR AAV2 targeting siRNA duplexes (positions 7999 to 9750), and includes siRNA duplexes with alternate chemistries. Of the 3′UTR_AAV2 targeting siRNA duplexes (positions 7999 to 9750) evaluated in the in vivo screen shown in Table 12, 1 achieved ≥80% knockdown of SCN9A, 4 achieved ≥60% knockdown of SCN9A, 6 achieved ≥30% knockdown of SCN9A, and 7 achieved ≥20% knockdown of SCN9A.

TABLE 12 Efficacy and duration of exemplary distal 3′UTR targeting SCN9A siRNAs (positions 7999 to 9750) in mice % Message Remaining at Day St. Treatment* Chemistry 14 post treatment Dev. PBS n/a 100.00 9.16 AAV-only 109.27 7.75 AD-64228.39 (AAV control) Fluoro 64.03 9.62 (TTR control) AD-802471.2 N6-C16 + VP + Fluoro 28.30 3.24 AD-961342.2 N6-C16 + VP + Non-Fluoro 42.46 6.81 AD-961334.2 N6-C16 + VP + Non-Fluoro 29.65 2.41 AD-1010697.2 N6-C16 + VP + Non-Fluoro 84.05 13.41 AD-1010698.2 N6-C16 + VP + Non-Fluoro 72.76 10.07 AD-802123.2 N6-C16 + VP + Fluoro 36.01 1.91 AD-801647.2 N6-C16 + VP + Fluoro 10.26 0.94 AD-961163.2 N6-C16 + VP + Fluoro + 61.24 15.34 GNA (*the number following the decimal point in a duplex name merely refers to a batch production number)

Table 19 and FIG. 2 (siRNA duplexes correspond to siRNA sequences in Table 18 and FIGS. 1A-1C) demonstrate the results of the in vivo screen for the ORF-1-targeting duplexes with the chemistries described in Table 18 and shown in FIGS. 1A-1C. Of the ORF-1 targeting duplexes evaluated in the in vivo screen shown in Table 19, 1 achieved ≥80% knockdown of SCN9A, 8 achieved ≥70% knockdown of SCN9A, 13 achieved ≥60% knockdown of SCN9A, 14 achieved ≥50% knockdown of SCN9A, and 15 achieved ≥30% knockdown of SCN9A. The results summarized in Table 19 also demonstrate that several modifications were tolerable in vivo with similar or improved potency compared to parent duplexes.

TABLE 19 Efficacy of exemplary SCN9A siRNA duplexes in mice. In this table, the column “Duplex Name” provides the numerical part of the duplex name without a suffix (e.g., number following the decimal point that can be included in a duplex name). The suffix merely refers to a batch production number. The suffix can be omitted from the duplex name without changing the chemical structure. For example, duplex AD-795305 in Table 19 refers to the same duplex as AD-795305.2 in Table 18. Duplex Name Day 14 post-treatment (Administered at 3 mg/kg) % SCN9A Message Remaining StDev PBS 100.00 4.16 AD-795305 (parent) 31.56 2.46 AD-1251249 23.56 3.45 AD-1251251 19.56 1.28 AD-1010663 (parent) 34.01 4.55 AD-1251301 64.01 6.65 AD-961179 (parent) 35.99 21.51 AD-1251317 36.50 6.98 AD-1251318 23.35 2.38 AD-1251323 33.32 5.62 AD-1251325 27.82 2.14 AD-795634 (parent) 25.39 5.54 AD-1251363 20.81 6.51 AD-1251364 26.37 6.88 AD-1251373 41.23 5.94 AD-1251385 29.05 12.32 AD-1251391 86.58 13.78

Following in vitro testing in Example 3 and the results in Table 17, a subset of duplexes were selected and placed in two groups: screen 1, which included AD-1010663.3, AD-1251301.1, AD-1251249.1, AD-1251251.1, AD-795305.3, AD-1251363.1, AD-1251364.1, AD-1251373.1, AD-795634.4, AD-1251385.1, AD-1251391.1, AD-1251317.1, AD-1251318.1, AD-1251323.1, AD-1251325.1, and AD-961179.3, screen 2 which included AD-1251492.1, AD-1251279.1, AD-961334.3, AD-1251284.1, AD-1251334.1, AD-1251377.1, AD-1251398.1, AD-1251399.1, AD-1251274.2, AD-961188.3, AD-1251411.1, AD-1251419.1, AD-796825, AD-1251428.1, AD-797564.4, and AD-1251434.1. The percent SCN9A message remaining when these duplexes were tested at a 0.1 nM (FIG. 4A), 1 nM (FIG. 4B), and 10 nM (FIG. 4C) was graphed versus the position in the target SCN9A mRNA of the sense strand of the tested duplex. From these graphs, a set of duplexes was selected for in vivo investigation, which are shown in Table 20 and FIGS. 3A-3D.

Table 21 and FIG. 5 (siRNA duplexes correspond to siRNA sequences in Table 20 and FIGS. 3A-3D) demonstrate the results of the in vivo screen for the duplexes targeting region 2 of the 3′ UTR of SCN9A (HsSCN9A_3 UTR2, e.g., positions 7999 to 9750), ORF-1 of SCN9A (HsSCN9A_ORF1rp, e.g., positions 299-2441), and ORF2 of SCN9A (HsSCN9A_ORF2rp, e.g., positions 2392-4345), with the chemistries described in Table 20 and shown in FIGS. 3A-3D. Of the exemplary duplexes investigated in the in vivo screen shown in Table 20, 4 achieved ≥80% knockdown of SCN9A, 11 achieved ≥70% knockdown of SCN9A, 13 achieved ≥50% knockdown of SCN9A, 14 achieved ≥30% knockdown of SCN9A, 15 achieved ≥20% knockdown of SCN9A, and 16 achieved ≥10% knockdown of SCN9A. The results summarized in Table 19 also demonstrate that several modifications were tolerable in vivo with similar potency compared to parent duplexes.

TABLE 21 Efficacy of exemplary SCN9A siRNA duplexes in mice. In this table, the exemplary duplexes investigated correspond to those summarized in Table 20 and FIGS. 3A-3D. The prior parent data corresponds to duplexes tested with data summarized in Tables 10-12. The column “Parent Duplex Name” provides the numerical part of the duplex name without a suffix (e.g., number following the decimal point that can be included in a duplex name). The suffix merely refers to a batch production number. The suffix can be omitted from the duplex name without changing the chemical structure. For example, duplex AD-802471 in Table 21 refers to the same duplex as AD-802471.2 in Table 12. Exemplary Duplexes Investigated Prior Parent Data (see, Tables 10-12) Duplex Name Day 14 post-treatment Day 14 post-treatment (Administered % SCN9A Message Parent Duplex % SCN9A Message AAV at 3 mg/kg) Remaining StDev Name Remaining StDev 3UTR2 PBS 100.00 17.39 AD-1251492.2* 19.42 13.58 AD-802471 28.30 3.24 AD-961334.2 (Parent) 17.15 1.96 AD-961334 (self) 29.65 2.41 AD-1251279.2 13.34 3.34 AD-961334 29.65 2.41 ORF1rp PBS 100.00 29.22 AD-1251284.2* 14.43 2.84 AD-1010661 41.08 6.19 AD-1251334.2* 65.74 28.11 AD-795366 29.15 4.74 AD-1251377.2* 28.53 17.61 AD-795634 20.80 6.50 AD-1251398.2* 81.26 6.35 AD-795913 26.74 5.61 AD-1251399.2* 75.63 37.43 AD-795913 26.74 5.61 AD-961188.2 (Parent) 44.91 20.18 AD-961188 (self) 38.51 0.36 AD-1251274.2 27.38 2.23 AD-961188 38.51 0.36 ORF2rp PBS 100.00 21.48 AD-796825.2 (Parent) 20.38 2.13 AD-796825 Self) 12.49 2.90 AD-1251411.2 24.57 5.44 AD-796825 12.49 2.90 AD-1251419.2 28.49 4.48 AD-796825 12.49 2.90 AD-797564.3 (Parent) 22.90 5.49 AD-797564 (self) 13.59 5.19 AD-1251428.2 43.00 7.98 AD-797564 13.59 5.19 AD-1251434.2 26.85 10.83 AD-797564 13.59 5.19 *parents not screened in this study 

We claim:
 1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of sodium channel, voltage gated, type IX alpha subunit (SCN9A), wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 and wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A 2B, 4A, 4B, 5A, 5B, 6A, 6B, 13A, 13B, 14A, 14B, 15A, 15B, 16, 18, and 20 that corresponds to the antisense sequence.
 2. The dsRNA agent of claim 1, wherein the portion of the sense strand is a portion within nucleotides 581-601, 760-780, or 8498-8518 of SEQ ID NO:
 4001. 3. The dsRNA agent of claim 1 or 2, wherein the portion of the sense strand is a portion within a sense strand from a duplex chosen from AD-1251284 (UGUCGAGUACACUUUUACUGA (SEQ ID NO:4827)), AD-961334 (CAACACAATUTCUUCUUAGCA (SEQ ID NO: 5026)), or AD-1251325 (AAAACAAUCUUCCGUUUCAAA (SEQ ID NO: 4822)).
 4. The dsRNA agent of any one of claims 1-3, wherein the portion of the sense strand is a sense strand chosen from the sense strands of AD-1251284 (UGUCGAGUACACUUUUACUGA (SEQ ID NO:4827)), AD-961334 (CAACACAATUTCUUCUUAGCA (SEQ ID NO: 5026)), or AD-1251325 (AAAACAAUCUUCCGUUUCAAA (SEQ ID NO: 4822)).
 5. The dsRNA of any one of claims 1-4, wherein the portion of the antisense strand is a portion within an antisense strand from a duplex chosen from AD-1251284 (UCAGTAAAAGUGUACTCGACAUU (SEQ ID NO: 5093)), AD-961334 (UGCUAAGAAGAAATUGUGUUGUU (SEQ ID NO: 5292)), or AD-1251325 (UUUGAAACGGAAGAUUGUUUUCC (SEQ ID NO: 5088)).
 6. The dsRNA of any one of claims 1-5, wherein the portion of the antisense strand is an antisense strand chosen the antisense strands of AD-1251284 (UCAGTAAAAGUGUACTCGACAUU (SEQ ID NO: 5093)), AD-961334 (UGCUAAGAAGAAATUGUGUUGUU (SEQ ID NO: 5292)), or AD-1251325 (UUUGAAACGGAAGAUUGUUUUCC (SEQ ID NO: 5088)).
 7. The dsRNA of any one of claims 1-6, wherein the sense strand and the antisense strand comprise nucleotide sequences of the paired sense strand and antisense strand of a duplex selected from AD-1251284 (SEQ ID NO: 4827 and 5093), AD-961334 (SEQ ID NO: 5026 and 5292), or AD-1251325 (SEQ ID NO: 4822 and 5088).
 8. The dsRNA agent of any one of claims 1-7, wherein the antisense strand comprises a nucleotide sequence of an antisense sequence listed in Table 16, and the sense strand comprises a nucleotide sequence of a sense sequence listed in Table 16 that corresponds to the antisense sequence.
 9. The dsRNA agent of any one of claims 1-8, wherein the dsRNA agent is AD-1251284, AD-961334, AD-1251325, AD-1331352, AD-1209344, or AD-1331350.
 10. The dsRNA agent of any one of claims 1-9, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties.
 11. The dsRNA agent of claim 10, wherein the lipophilic moiety is conjugated via a linker or carrier.
 12. The dsRNA agent of claim 10 or 11, wherein one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand.
 13. The dsRNA agent of claim 12, wherein the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via a linker or carrier.
 14. The dsRNA agent of any one of claims 10-13, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.
 15. The dsRNA agent of claim 14, wherein the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.
 16. The dsRNA agent of any one of claims 10-15, wherein the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region.
 17. The dsRNA agent of any one of claims 10-15, wherein the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.
 18. The double-stranded iRNA agent of any one of claims 10-16, wherein the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.
 19. The dsRNA agent of any of the preceding claims, wherein the dsRNA agent comprises at least one modified nucleotide.
 20. The dsRNA agent of claim 19, wherein no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand are unmodified nucleotides.
 21. The dsRNA agent of claim 19, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.
 22. The dsRNA agent of any one of claims 19-21, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxythimidine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a glycol modified nucleotide, and a 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof.
 23. The dsRNA agent of any of the preceding claims, wherein at least one strand comprises a 3′ overhang of at least 2 nucleotides.
 24. The dsRNA agent of any of the preceding claims, wherein the double stranded region is 15-30 nucleotide pairs in length.
 25. The dsRNA agent of claim 24, wherein the double stranded region is 17-23 nucleotide pairs in length.
 26. The dsRNA agent of any of the preceding claims, wherein each strand has 19-30 nucleotides.
 27. The dsRNA agent of any of the preceding claims, wherein the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.
 28. The dsRNA agent of any one of claims 10-27, further comprising a targeting ligand, e.g., a ligand that targets a CNS tissue.
 29. The dsRNA agent of claim 28, wherein the targeting ligand is a ligand that targets a CNS tissue.
 30. The dsRNA agent of claim 29, wherein the CNS tissue is a brain tissue or a spinal tissue.
 31. The dsRNA agent of any one of the preceding claims, further comprising a phosphate or phosphate mimic at the 5′-end of the antisense strand.
 32. The dsRNA agent of claim 31, wherein the phosphate mimic is a 5′-vinyl phosphonate (VP).
 33. The dsRNA of any one of the preceding claims, wherein: (i) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 4029, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4295; (ii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 4228, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 4494; (iii) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 5339, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 5355; (iv) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 5800, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 5801; (v) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 5526, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO: 5681; or (vi) the sense strand comprises the sequence and all the modifications of SEQ ID NO: 5542, and the antisense strand comprises the sequence and all the modifications of SEQ ID NO:
 5697. 34. A cell containing the dsRNA agent of any one of claims 1-33.
 35. A pharmaceutical composition for inhibiting expression of a SCN9A, comprising the dsRNA agent of any one of claims 1-33.
 36. A method of inhibiting expression of SCN9A in a cell, the method comprising: (a) contacting the cell with the dsRNA agent of any one of claims 1-33, or a pharmaceutical composition of claim 35; and (b) maintaining the cell produced in step (a) for a time sufficient to reduce levels of SCN9A mRNA, SCN9A protein, or both of SCN9A mRNA and protein, thereby inhibiting expression of SCN9A in the cell.
 37. The method of claim 36, wherein the cell is within a subject.
 38. The method of claim 37, wherein the subject is a human.
 39. The method of claim 38, wherein the subject has been diagnosed with a SCN9A-associated disorder, e.g., pain, e.g., chronic pain e.g., inflammatory pain, neuropathic pain, pain hypersensitivity, pain hyposensitivity, primary erythromelalgia (PE), paroxysmal extreme pain disorder (PEPD), small fiber neuropathy (SFN), trigeminal neuralgia (TN), and pain associated with, e.g., cancer, arthritis, diabetes, traumatic injury and viral infections.
 40. A method of treating a subject having or diagnosed with having a SCN9A-associated disorder comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-33 or a pharmaceutical composition of claim 35, thereby treating the disorder.
 41. The method of claim 40, wherein the SCN9A-associated disorder is pain, e.g., chronic pain.
 42. The method of claim 40, wherein the SCN9A-associated disorder is chronic pain.
 43. The method of claim 41 or 42, wherein the chronic pain is associated with one or more of the disorders in the group consisting of pain hypersensitivity, pain hyposensitivity, inability to sense pain, primary erythromelalgia (PE), paroxysmal extreme pain disorder (PEPD), small fiber neuropathy (SFN), trigeminal neuralgia (TN), or pain associated with cancer, arthritis, diabetes, traumatic injury or viral infections.
 44. The method of any one of claims 40-43, wherein treating comprises amelioration of at least one sign or symptom of the disorder.
 45. The method of any one of claims 40-44, wherein the treating comprises (a) reducing pain; or (b) inhibiting or reducing the expression or activity of SCN9A.
 46. The method of any one of claims 37-45, wherein the dsRNA agent is administered to the subject intracranially or intrathecally.
 47. The method of claim 44, wherein the dsRNA agent is administered to the subject intrathecally, intraventricularly, or intracerebrally.
 48. The method of any one of claims 37-47, further comprising administering to the subject an additional agent or therapy suitable for treatment or prevention of an SCN9A-associated disorder (e.g., non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen, opioids, or corticosteroids, acupuncture, therapeutic massage, dorsal root ganglion stimulation, spinal cord stimulation, or topical pain relievers). 